Methods of treating tuberous sclerosis complex with cannabidiol and everolimus

ABSTRACT

The present disclosure provides methods of treating tuberous sclerosis complex comprising administering cannabidiol and everolimus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 62/982,506, filed Feb. 27, 2020, and GB Provisional Application No. 2002754.6, filed Feb. 27, 2020, the entire contents of each of which are herein incorporated by reference in its entirety for all purposes.

BACKGROUND

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by growth of distinctive benign tumors (hamartomas) and malformations (hamartias) in multiple organ systems. Tumors of the central nervous system (CNS) are the leading cause of morbidity and mortality in TSC patients, followed by renal disease. Patients with TSC can suffer from abnormalities of the brain that may include seizures, intellectual disability, and developmental delay, as well as abnormalities of the skin, lung, kidneys, and heart. The disorder affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns.

Inherited defects or de novo mutations associated with TSC occur on one of two genes, TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present. The TSC1 gene, on chromosome 9, produces a protein called hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein tuberin. Scientists believe these proteins act in a complex as growth suppressors by inhibiting the activation of a master, evolutionarily conserved kinase called mTOR. Loss of regulation of mTOR occurs in cells lacking either hamartin or tuberin, and this leads to abnormal differentiation and development, and to the generation of enlarged cells, as are seen in TSC brain lesions.

Between one fourth and one half of all children with TSC develop autism spectrum disorders (ASD). Almost all patients with TSC also develop epilepsy and most develop multiple seizure types. Furthermore, most patients with TSC have refractory epilepsy, and therefore are resistant to treatment with one or more anti-epileptic drugs. On average, TSC patients are resistant to five AED.

Presently, there is no monotherapy that can treat all conditions and symptoms associated with TSC. In order to treat tumors and epileptic seizures, TSC patients are commonly administered combinations of the drugs, such as an mTor inhibitor (e.g., everolimus) and an antiepileptic drug. In addition to TSC being generally refractory to multiple antiepileptic drugs, antiepileptic drugs can also have an adverse drug-drug interaction (“DDI”) with everolimus. Thus, there exists a need for a safe and effective method of treating patients with everolimus and an antiepileptic drug.

SUMMARY

Applicant has discovered a method of safely and effectively treating TSC patients with cannabidiol (“CBD”) and everolimus.

In some embodiments, the disclosure provides a method of treating TSC in a patient in need thereof, comprising administering everolimus in combination with a reduced dose of CBD.

In some embodiments, the patient is administered from about 2 mg to about 10 mg of everolimus. In some embodiments, the patient is administered about 2.5 mg, about 5 mg, or about 10 mg of everolimus. In some embodiments, the patient is administered 2.5 mg or 5 mg of everolimus. In some embodiments, the patient is administered 2.5 mg of everolimus. In some embodiments, the patient is administered 5 mg of everolimus.

In some embodiments, the reduced dose of CBD is at least about 10% less than the dose of CBD the patient would otherwise receive in the absence of everolimus. In some embodiments, the reduced dose of CBD is between 10% to 90% less than the dose of CBD the patient would otherwise receive in the absence of everolimus. In some embodiments, the reduced dose of CBD ranges from about 5 mg/kg/day to about 20 mg/kg/day. In some embodiments, the reduced dose of CBD is about 5 mg/kg/day, about 7 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 12 mg/kg/day, about 14 mg/kg/day, about 15 mg/kg/day, about 16 mg/kg/day, about 18 mg/kg/day, or about 20 mg/kg/day. In some embodiments, the reduced dose of CBD is about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, or about 20 mg/kg/day. In some embodiments, the reduced dose of CBD is about 5 mg/kg/day. In some embodiments, the reduced dose of CBD is about 10 mg/kg/day. In some embodiments, the reduced dose of CBD is about 15 mg/kg/day. In some embodiments, the reduced dose of CBD is about 20 mg/kg/day.

In some embodiments, the patient is administered 2.5 mg of everolimus, and a reduced dose of CBD selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day. In some embodiments, the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day. In some embodiments, the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 20 mg/kg/day. In some embodiments, the patient is administered 5 mg of everolimus, and a reduced dose of CBD selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day. In some embodiments, the patient is administered 5 mg of everolimus, and the reduced dose of CBD selected from the group consisting of about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day. In some embodiments, the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 20 mg/kg/day.

In some embodiments, the CBD is present in a highly purified botanical drug substance. In some embodiments, the CBD has a purity of at least 95% w/w. In some embodiments, the CBD has a purity of at least 98% w/w. In some embodiments, wherein the CBD is synthetic CBD.

In some embodiments, the disclosure provides for methods of treating seizures associated with tuberous sclerosis complex in an everolimus-naive patient, wherein the patient is currently treated with CBD, comprising: (a) administering everolimus to the patient; (b) monitoring the patient's blood plasma levels of everolimus; and (c) if the patient's blood plasma trough concentrations of everolimus exceed 15 ng/mL, then reducing the dose of everolimus by at least 10%. In some embodiments, the patient is treated with 25 mg/kg/day CBD. In some embodiments, step (a) comprises administering 5 mg/m² everolimus once daily. In some embodiments, step (a) comprises administering a starting dose of everolimus of 5 mg/m² everolimus once daily, and titrating up the everolimus dose by no more than 5 mg (e.g., 1, 2, 3, 4, or 5 mg). In some embodiments, the dose of everolimus is titrated every 1 to 2 weeks until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL. In some embodiments, the monitoring in step (b) occurs 1 to 2 weeks after the patients begins administering everolimus. In some embodiments, the dose of everolimus is reduced to provide blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL. In some embodiments, the dose of everlimus is reduced (e.g., by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%) to provide a Cmax that is not more than about 45 ng/mL, about 40 ng/mL, about 35 ng/mL, about 30 ng/mL, about 25 ng/mL or about 20 ng/mL. In some embodiments, the dose of everlimus is reduced to provide an AUC₀₋₂₄ that is not more than about 490 h*ng/mL, about 450 h*ng/mL, about 400 ng/mL, about 350 h*ng/mL, about 300 h*ng/mL, about 250 h*ng/mL, or about 200 h*ng/mL.

In some embodiments, the disclosure provides for methods of treating seizures associated with tuberous sclerosis complex in an everolimus-naive patient, wherein the patient is currently treated with CBD, comprising: (a) administering everolimus to the patient; (b) monitoring the patient's blood plasma levels of everolimus; and (c) if the patient's Cmax exceeds 50 ng/mL or AUC_(0-last) 500 h*ng/mL, then reducing the dose of everolimus by at least 10%. In some embodiments, the patient is treated with 25 mg/kg/day CBD. In some embodiments, step (a) comprises administering 5 mg/m² everolimus once daily. In some embodiments, step (a) comprises administering a starting dose of everolimus of 5 mg/m² everolimus once daily, and titrating up the everolimus dose by no more than 5 mg (e.g., 1, 2, 3, 4, or 5 mg). In some embodiments, the dose of everolimus is titrated every 1 to 2 weeks until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL. In some embodiments, the monitoring in step (b) occurs 1 to 2 weeks after the patients begins administering everolimus. In some embodiments, the dose of everolimus is reduced to provide blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL. In some embodiments, the dose of everlimus is reduced (e.g., by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%) to provide a Cmax that is not more than about 45 ng/mL, about 40 ng/mL, about 35 ng/mL, about 30 ng/mL, about 25 ng/mL or about 20 ng/mL. In some embodiments, the dose of everlimus is reduced to provide an AUC₀₋₂₄ that is not more than about 490 h*ng/mL, about 450 h*ng/mL, about 400 ng/mL, about 350 h*ng/mL, about 300 h*ng/mL, about 250 h*ng/mL, or about 200 h*ng/mL.

In some embodiments, the seizures are focal seizures. In some embodiments, the focal seizures are focal motor seizures without impairment of consciousness or awareness; focal seizures with impairment of consciousness or awareness; or focal seizures evolving to bilateral generalized convulsive seizures and generalized seizures. In some embodiments, the generalized seizures are tonic-clonic, tonic, clonic or atonic seizures. In some embodiments, the seizures are generalized seizures. In some embodiments, the generalized seizures are tonic-clonic, tonic, clonic or atonic seizures. In some embodiments, the total number of seizures are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to baseline. In some embodiments, the total number of seizures are reduced by at least 50% compared to baseline. In some embodiments, the total number of focal seizures are reduced by at least 50% compared to baseline. In some embodiments, the focal seizures are focal motor seizures without impairment of consciousness or awareness; focal seizures with impairment of consciousness or awareness; or focal seizures evolving to bilateral generalized convulsive seizures and generalized seizures. In some embodiments, the total number of tonic-clonic, tonic, clonic, atonic or absence seizures are reduced by at least 50% compared to baseline.

In some embodiments, the disclosure provides a method of treating seizures associated with tuberous sclerosis complex in a patient in need thereof, comprising administering from 2.5-10 mg of everolimus in combination with cannabidiol (CBD), wherein the CBD has a purity of at least 95% w/w, and the CBD is administered at a dose ranging from about 10 mg/kg/day to about 20 mg/kg/day. In some embodiments, the patient is administered about 2.5 mg, about 5 mg, or about 10 mg of everolimus. In some embodiments, the patient is administered 2.5 mg or 5 mg of everolimus. In some embodiments, the dose of CBD is about 10 mg/kg/day, about 15 mg/kg/day, or about 20 mg/kg/day. In some embodiments, the dose of CBD is about 20 mg/kg/day. In some embodiments, the seizures are focal seizures. In some embodiments, the focal seizures are focal motor seizures without impairment of consciousness or awareness; focal seizures with impairment of consciousness or awareness; or focal seizures evolving to bilateral generalized convulsive seizures and generalized seizures. In some embodiments, the generalized seizures are tonic-clonic, tonic, clonic or atonic seizures. In some embodiments, the seizures are generalized seizures. In some embodiments, the generalized seizures are tonic-clonic, tonic, clonic or atonic seizures. In some embodiments, the total number of seizures are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to baseline. In some embodiments, the total number of seizures are reduced by at least 50% compared to baseline. In some embodiments, the total number of focal seizures are reduced by at least 50% compared to baseline. In some embodiments, the focal seizures are focal motor seizures without impairment of consciousness or awareness; focal seizures with impairment of consciousness or awareness; or focal seizures evolving to bilateral generalized convulsive seizures and generalized seizures. In some embodiments, the total number of tonic-clonic, tonic, clonic, atonic or absence seizures are reduced by at least 50% compared to baseline.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically illustrates that trial design to measure the impact of everolimus (5 mg daily dose) on the pharmacokinetics of CBD (25 mg/kg/day).

FIG. 2 shows the blood concentration profile (ng/mL) of everolimus in patients that received everolimus alone, and patients that received both everolimus and CBD.

FIG. 3 shows the blood concentration profile (ng/mL) of CBD in patients that received CBD alone, and patients that received both everolimus and CBD.

FIG. 4 shows the blood concentration profile (ng/mL) of 1-OH-CBD in patients that received CBD alone, and patients that received both everolimus and CBD.

FIG. 5 shows the blood concentration profile (ng/mL) of 1-COOH-CBD in patients that received CBD alone, and patients that received both everolimus and CBD.

DETAILED DESCRIPTION Definitions

As used herein, the term “about” refers to an acceptable degree of variation in the art. In some embodiments, “about” means plus or minus 10% of the referenced number unless otherwise stated or otherwise evident by the context, and except where such a range would exceed 100% of a possible value, or fall below 0% of a possible value, such as less than 0% content of an ingredient, or more than 100% of the total contents of a composition.

The term “a” or “an” refers to one or more of that entity; for example, “an antiepileptic drug” (AED) refers to one or more AED or at least one AED. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

In some embodiments, the compositions contain cannabidiol (“CBD”). As used herein, CBD refers to synthetic CBD or to a Cannabis extract, which contains between 95% w/w to 100% w/w CBD. In some embodiments, the CBD extract can contain between about 95% w/w, about 96% w/w, about 97% w/w, about 98% w/w, about 99% w/w, or about 100% w/w. In some embodiments, the CBD extract contains 98% w/w CBD. Such a Cannabis extract, containing at least 95% w/w CBD, may be referred to as a highly pure botanical drug substance. In some embodiments, the CBD extract contains about 99% w/w CBD. Unless otherwise noted, references to CBD in the present disclosure should be understood as references to the decarboxylated version of the CBD, and do not include the acidic CBDA.

As used herein, “purity” and “pure” when modifying CBD refers to the weight of CBD in the drug substance based on the total weight of all cannabinoids in the drug substance. Additional cannabinoids that may be present in the drug substance include, without limitation, CBDV, Δ9THC, and CBD-C4, and combinations thereof. For avoidance of doubt, drug substance is distinct from a drug product. Drug product refers to the pharmaceutical composition containing the drug substance. Accordingly, when the present disclosure refers to administering CBD which has a purity of at least 95% w/w (or similar expressions), the purity refers to CBD in the drug substance, not the drug product.

As defined herein, a “reduced dose” of CBD is less than the dose that an otherwise identical patient with TSC would receive if said patient were not coadministered everolimus. In other words, the reduced dose of CBD administered to a patient with TSC in combination with everolimus is less than the dose of CBD administered to the same (or similar) TSC patient in the absence of everolimus. The dose of CBD that the patient would otherwise receive in the absence of everolimus (i.e., if the patient was not coadministered everolimus) may be referred to as the “reference dose” or the “recommended dose” of CBD. “Reference dose” or “recommended dose” may be used interchangeably. Furthermore, “reference dose” and “recommended dose” are distinct from a “starting dose.” A “starting dose” is the dose administered to the patient to initiate treatment. The “starting dose” is a lower dose than the “reference dose” and is administered for a period of time (e.g., over the course of 1 week) before the patient increases the dose to the “reference dose”.

In some embodiments, the “reference dose” or “recommended dose” of CBD ranges from about 10 mg/kg/day, to about 50 mg/kg/day. In some embodiments, the “reference dose” of CBD is the dose that is approved (e.g., by the FDA or the EMA) for treating seizures in the particular patient. For nearly all FDA approved drugs, the “recommended” dose (or doses) of the drug are determined based on the plasma level (or range of plasma levels) of the drug required to provide the desired clinical effect(s) and/or avoid undesirable side effects. The recommended dose(s) of a particular drug are those recognized in the art as suitable for treating a patient with particular physical characteristics (or within a range of particular characteristics), and are thus the dose(s) provided in the package insert for the drug. There may be multiple recommended doses for a particular condition. For example, there are two recommended doses of EPIDIOLEX® (November 2018; revised July 2020) for treating seizures associated with Lennox-Gastaut or Dravet Syndrome—(1) a maintenance dosage of 10 mg/kg/day and (2) a maximum recommended maintenance dosage of 20 mg/kg/day. For avoidance of doubt, both doses (i.e. (1) and (2)) are considered a “recommended dose” according to this disclosure. Thus, in various embodiments, the methods of the present disclosure are directed to adjustments or changes in the dosing of CBD relative to the FDA “recommended” dose, e.g., in the package insert for CBD, as suitable for treating a TSC patient with particular physical characteristics. The particular characteristics may include hepatic impairment, since patients with moderate or severe hepatic impairment receive different recommended doses of CBD as described herein and according to the EPIDIOLEX® drug label (November 2018; revised July 2020). Thus, as used herein, the “reference dose” or “recommended dose” for CBD is distinct from doses which may be disclosed by particular physicians for particular patients. Depending on the specific pharmacokinetics and pharmacodynamics of the drug, the recommended dose may vary depending on one or more physical and physiological characteristics of the patient, for example age, gender, weight, body mass index, liver metabolic enzyme status (e.g., poor or extensive metabolizer status), disease state, etc.

At the time of this disclosure, Applicant contemplates that the approved dosing of CBD (in the absence of everolimus) for TSC ranges from 5 mg/kg/day to 25 mg/kg/day (e.g., for patients with normal hepatic status or mild hepatic impairment). Applicant contemplates that TSC patients will receive a starting dose of 5 mg/kg/day of CBD, and the starting dose may be increased to achieve and/or maintain efficacy. Typically, efficacious doses may be 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, or about 25 mg/kg/day when administered in the absence of everolimus, although the exact efficacious dose will depend on the specific patient and the patient's physical and physiological characteristics. Therefore, in some embodiments, the “reference dose” of CBD is about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, or about 25 mg/kg/day. In July of 2020 (after the priority date of the present application), the FDA approved CBD for treating seizures associated with TSC. According to the drug label for EPIDIOLEX® (revised July 2020), the recommended dosage for treating seizures associated with is 25 mg/kg/day (e.g., for patients with normal hepatic status or mild hepatic impairment). Thus, in some embodiments, the reduced dose is less than the 25 mg/kg/day reference dose of EPIDIOLEX® that is approved for treating seizures associated TSC according to the drug label for EPIDIOLEX® (revised July 2020).

In some embodiments, the methods of the present disclosure provide for reducing the dose of CBD that the patient would otherwise receive in the absence of everolimus. For example, if a TSC patient is administered 10 mg/kg/day of CBD in the absence of everolimus, when coadministered everolimus the TSC patient will receive less than 10 mg/kg/day as a reduced dose (e.g. about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, or about 9 mg/kg/day). For example, if a TSC patient is administered 15 mg/kg/day of CBD in the absence of everolimus, when coadministered everolimus the TSC patient will receive less than 15 mg/kg/day as a reduced dose (e.g. e.g. about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, or about 14 mg/kg/day). As another example, if a TSC patient is administered 20 mg/kg/day of CBD in the absence of everolimus, when coadministered everolimus the TSC patient will receive less than 20 mg/kg/day as a reduced dose (e.g. about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day). As yet another example, if a TSC patient is administered 25 mg/kg/day of CBD in the absence of everolimus, when coadministered everolimus the TSC patient will receive less than 25 mg/kg/day as a reduced dose (e.g. about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day, about 20 mg/kg/day, about 21 mg/kg/day, about 22 mg/kg/day, about 23 mg/kg/day, or about 24 mg/kg/day). As used herein, the amounts of CBD in mg/kg/day or in mg/kg refer to the amount of drug substance (i.e., CBD) that is administered regardless of the purity of CBD in the drug sub stance.

As used herein, “treat” or “treating” means one or more of relieving, alleviating, delaying, reducing, reversing, improving, or managing at least one symptom of a condition in a subject. Such symptoms may include, without limitation, the onset, frequency, or duration of one or more types of seizures. Seizures associated with TSC include, without limitation, infantile seizures, focal seizures (with or without impairment or consciousness or awareness; focal seizures evolving to bilaterial generalized convulsive seizures and generalized seizures) tonic seizures, clonic seizures, tonic-clonic seizures, atonic seizures, myoclonic seizures, and absence seizures. The term “treating” may also mean one or more of arresting, delaying the onset (i.e., the period prior to clinical manifestation of the condition) or reducing the risk of developing or worsening a condition.

As used herein, the term an “otherwise identical patient” or an “otherwise identical patient who is not coadministered everolimus” refers to a patient whose physical characteristics relevant to drug (e.g., CBD) dosing are expected to be substantially the same as that of the patient being treated with a reduced CBD dose according to the presently disclosed methods—except that the patient is not concomitantly administered everolimus. In some embodiments, the otherwise identical patient will be of substantially the same age, sex, and body weight. In some embodiments, the substantially identical patient will also have substantially identical hepatic function and drug metabolism.

As used herein, the term “coadministered” means that one or more drugs are administered on the same day. In some embodiments, the one or more drugs (e.g. cannabidiol and everolimus) are administered sequentially. In some embodiments, the one or more drugs (e.g. cannabidiol and everolimus) are administered simultaneously.

As used herein, “hepatic impairment” means a reduction in normal liver executory and metabolic function compared to an otherwise healthy liver. The liver is involved in the clearance of many drugs through a variety of oxidative and conjugative metabolic pathways and/or through biliary excretion of unchanged drug or metabolites. Alterations of these excretory and metabolic activities by hepatic impairment can lead to drug accumulation or, less often, failure to form an active metabolite. In some embodiments, hepatic impairment can be determined using the Child Pugh score. The Child Pugh score is described in Cholongitas, et al. “Systematic review: The model for end-stage liver disease—should it replace Child-Pugh's classification for assessing prognosis in cirrhosis?”. Alimentary Pharmacology & Therapeutics. 22 (11-22): 1079-89, which is herein incorporated by reference in its entirety.

The Child Pugh score employs five clinical measures of liver disease. Each measure is scored 1-3, with 3 indicating most severe derangement. Either the prothrombin time or INR should be used to calculate the Child-Pugh score, not both.

Measure 1 point 2 points 3 points Total bilirubin, (mg/dL) (<2) (2-3) (>3) Serum albumin, g/dL >3.5 2.8-3.5 <2.8 Prothrombin time, <4.0 4.0-6.0 >6.0 prolongation (s) INR <1.7 1.7-2.3 >2.3 Ascites None Mild (or Moderate to suppressed severe with medication) (or refractory) Hepatic encephalopathy None Grade I-II Grade III-IV

Chronic liver disease is classified into Child-Pugh class A to C, employing the added score from above.

Points Class 5-6 A 7-9 B 10-15 C

In some embodiments, a patient with “mild hepatic impairment” has a Child Pugh score of A. In some embodiments, a patient with “moderate hepatic impairment” has a Child Pugh score of B. In some embodiments, a patient with “severe hepatic impairment” has a Child Pugh score of C.

In some embodiments, “mild hepatic impairment” is bilirubin≤1×the upper limit of the normal range (“ULN”) and aspartate aminotransferase (“AST”)>1×ULN, or bilirubin>1.0-1.5×ULN and any amount of AST above ULN is present. In some embodiments, “moderate hepatic impairment” is bilirubin>1.5-3.033×ULN and any amount of AST above ULN is present. In some embodiments, “severe hepatic impairment” is bilirubin≥3.0×ULN and any amount of AST above ULN is present. In some embodiments, serum transaminases (ALT and AST) and total bilirubin levels are obtained prior to starting treatment.

Methods of Treating TSC Using CBD and Everolimus

Patients with TSC have treatment-resistant epilepsy. This form of epilepsy is associated with several seizure types including, without limitation, infantile seizures, focal seizures (with or without impairment or consciousness or awareness; focal seizures evolving to bilaterial generalized convulsive seizures and generalized seizures) tonic seizures, clonic seizures, tonic-clonic seizures, atonic seizures, myoclonic seizures, and absence seizures, tonic seizures, myoclonic seizures, and absence seizures. Patients with TSC also exhibit noncancerous growths in many parts of the body, including the brain, skin, kidneys, and other organs. The present disclosure provides methods of treating seizures in patients diagnosed with TSC, comprising administering everolimus in combination with a reduced dose of CBD.

CBD (under the brandname EPIDIOLEX®) is approved by the Food and Drug Administration (FDA) for the treatment of Lennox-Gastaut syndrome and Dravet Syndrome at doses ranging from 5 mg/kg/day to 20 mg/kg/day. Everolimus is approved by the FDA for the treatment of patients with TSC that have subependymal giant cell astrocytoma (SEGA) at a dose of 4.5 mg/m² once daily, for TSC-associated renal angiomyolipoma at a dose of 10 mg orally once daily, and as an adjunctive treatment for TSC-associated partial-onset seizures at a dose of 5 mg/m² once daily.

Because TSC patients also experience seizures described herein, Applicant discovered that CBD can be used in combination with everolimus to treat TSC patients. Indeed, in July of 2020, after the priority date of the present application, the FDA approved CBD for treating seizures associated with TSC. According to the drug label for EPIDIOLEX® (revised July 2020), the recommended dosage for treating seizures associated with is 25 mg/kg/day (e.g., for patients with normal hepatic status or mild hepatic impairment). However, Applicant discovered a potentially serious adverse drug-drug interaction (“DDI”) between CBD and everolimus. Applicant found that reducing the dose of everolimus (e.g., as prescribed in the everolimus package insert or drug label) is not sufficient to address the DDI between CBD and everolimus. In some embodiments, Applicant surprisingly and unexpectedly found that the reference dose of CBD must be reduced to safely treat TSC patients that are concurrently treated with everolimus, and the reduced dose is also efficacious. In some embodiments, Applicant surprisingly and unexpectedly found that reducing the dose of both CBD and everolimus is critical to safely and effectively coadminister CBD and everolimus in order to treat TSC.

The most common types of DDIs involve the inhibition or induction of one or more drug-metabolizing enzymes by a drug. When inhibitors or inducers of a particular drug-metabolizing enzyme are coadministered with a drug that is metabolized by that enzyme (known as a substrate of that enzyme), the pharmacokinetic parameters of one or both drugs change, leading to increased or decreased drug exposures. It is this change in exposure that may result in adverse events. However, an increase in pharmacokinetic parameters, alone, does not necessarily produce an adverse event, and require a dose modification. Only if the increase in the pharmacokinetic parameters presents a risk to the patient is a dose modification required. However, dosing modifications are unpredictable. Reducing a dose too much risks undertreating the patients, but over-dosing (e.g. not reducing the dose, or not reducing the dose enough) can increases exposure to potentially dangerous levels.

A drug of interest is classified as a weak, moderate, or strong CYP3A inhibitor based on the effect of the drug of interest on the plasma area under-the-curve (AUC) of the prototypical CYP3A4/CYP3A5 substrate midazolam. If a drug of interest increases the AUC of midazolam≥5 fold, the drug of interest is a strong CYP3A inhibitor. If the drug of interest increases the AUC of midazolam increases >2.0 to 4.9 fold, the drug of interest is a moderate CYP3A inhibitor. If the drug of interest increases the AUC of midazolam≤2 fold, the drug of interest is a weak CYP3A inhibitor.

According to the FDA-approved drug label (also referred to as the Prescribing Information) for everolimus (AFINITOR® and AFINITOR DISPERZ®, updated January 2019, herein incorporated by reference in its entirety), everolimus is a substrate for P-gp and CYP3A4. Everolimus is also reported to be a substrate for CYP3A5, and CYP2C8.

Combined P-pg and CYP3A4 inhibitors are known to increase everolimus exposure. For example, ketoconazole, a P-gp and strong CYP3A4 inhibitor, increases the C_(max) and AUC of everolimus by 3.9 and 15-fold, respectively. Erythromycin, a P-gp and moderate CYP3A4 inhibitor increases the C_(max) and AUC of everolimus by 2.0 and 4.4-fold, respectively. Verapamil, a P-gp and moderate CYP3A4 inhibitor, increases the C_(max) and AUC of everolimus by 2.3 and 3.5-fold, respectively.

The FDA label of everolimus contraindicates concomitant administration of everolimus and a P-gp and strong CYP3A4 inhibitors. With respect to P-gp and moderate CYP3A4 inhibitors, the everolimus label instructs that the dose of everolimus is reduced. Specifically, for patients with TSC-associated renal angiomyolipoma, the label instructs patients to reduce the dose of everolimus to 2.5 mg once daily. The label also provides instructions to increase the dose to 5 mg if it can be tolerated. Three days after discontinuing the P-gp and moderate CYP3A4 inhibitor, patients can resume treatment with the everolimus dose that was administered prior to inhibitor use. In patents with TSC-associated SEGA and TSC-associated partial-onset seizures, the label instructs patients to reduce the daily dose (as described herein and on the FDA label) by 50%. If the reduced dose is lower than the lowest available strength of everolimus, patients should change to dosing every other day. Three days after discontinuing the P-gp and moderate CYP3A4 inhibitor, patients can resume treatment with the everolimus dose that was administered prior to inhibitor use.

The label also indicates that everolimus shows low levels of CYP3A4 of CYP2D6 inhibition, but the interaction was deemed “not clinically significant.” (Section 12.3 of the AFINITOR/AFINITOR DISPERZ Label). Specifically, coadministration of an oral dose of midazolam (sensitive CYP3A4 substrate) with AFINITOR resulted in a 25% increase in midazolam Cmax and a 30% increase in midazolam AUC_(0-inf). Similarly, co-administration of everolimus with other CYP3A4 substrates, including atorvastatin, pravastatin, and simvastatin, did not produce pharmacodynamic result and was demeaned not clinically relevant. Thus, prior to the present disclosure, everolimus was not expected cause clinically significant drug-drug interactions through CYP3A4 of CYP2D6 inhibition. Accordingly, there was no expectation that the dosage of a drug coadminstered with everolimus, such as CBD, should be reduced in order to safely treat TSC patients.

The pharmacological properties of CBD are not fully elucidated. As indicated on the FDA approved label for EPIDIOLEX®, CBD is metabolized in the liver and the gut, and is a substrate CYP2C19 and CYP3A4 enzymes and UGT1A7, UGT1A9, and UGT2B7 isoforms. CBD inhibits uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes UGT1A9 and UGT2B7. CBD is reported to be an inhibitor of CYP2B6, CYP2C8, CYP2C9, and CYP2C19. Data also suggest that CBD has the potential to inhibit CYP3A4, but the strength or clinical significance of this interaction is unknown. CBD also has the potential to induce or inhibit CYP1A2 and CYP2B6 at clinically relevant concentrations, but the strength or clinical significance of this interaction is unknown. Thus, it is not known if CBD is a strong, moderate, or weak inhibitor of any CYP isoform, or indeed if CBD-mediated inhibition of any of any of these enzymes produces a clinically significant result. Lastly, Applicant discovered that a metabolite of CBD, 7-COOH-CBD is a substrate for P-gp. 7-COOH-CBD is an inhibitor of transport mediated via BCRP and BSEP at clinically relevant concentrations.

Applicant discovered that elevated levels of CBD cause transaminase elevation, rash, somnolence, sedation, lethargy, diarrhea, pyrexia, weight decreased, nasopharyngitis, irritability, oropharyngeal pain, and decreased appetite. Transaminase can lead to hepatic dysfunction, including unexplained nausea, vomiting, right upper quadrant abdominal pain, fatigue, anorexia, or jaundice and/or dark urine. Elevated exposure of everolimus exposure is known to cause non-infectious pneumonitis, infections, severe hypersensitivity to reactions, angioedema, stomatitis, renal failure, impaired wound healing, metabolic disorders, myelosuppression, risk of infection or reduced immune response with vaccination, embryo-fetal toxicity, and stomatis, respiratory tract infection. The methods of the disclosure reduce the incidence of one or more of the above side effects.

Applicant found that when CBD is administered at a steady state orally in combination with midazolam, CBD exerts no clinically significant effect on midazolam's pharmacokinetic parameters (See Example 2). In other words, the DDI with midazolam (if any) predicts that CBD is a weak inhibitor of CYP3A4. Therefore, CBD should have no clinically relevant impact on everolimus (a CYP3A4 substrate drug). Similarly, DDI studies for everolimus described herein predict that everolimus should have no clinically relevant impact on CBD. However, Applicant unexpectedly found that a potentially clinically significant DDI occurs when everolimus and CBD are co-administered. Without being bound by theory, the DDI that the Applicant observed may result from a previously unknown mechanism whereby CBD inhibits P-gp-mediated efflux of everolimus. The everolimus label contains no instructions to adjust the dose when administered with a P-gp inhibitor that is not also a moderate or strong CYP3A4 inhibitor. Additionally or alternatively, combined CYP3A4 inhibition by both CBD and everolimus may be significant enough to produce a new and unexpected pharmacodynamic effect.

Prior to Applicant's invention, it was believed that administering everolimus according to the instructions on the FDA-approved label was sufficient to address DDI. Because the everolimus label contains no instructions to adjust the dose when administered with a P-gp inhibitor that is not a moderate or strong CYP3A4 inhibitor, a skilled artisan would have believed that no dose reduction was required with treating a patient with both CBD and everolimus. For combined P-gp and moderate CYP3A4 inhibitors (which CBD is not), the everolimus label instructions patients to reduce the dose of everolimus from 10 mg, to a dose of 5 mg and then 2.5 mg. However, Applicant was surprised to discover that for patients already treated with everolimus at the time of initiating CBD treatment, the adjustment is not sufficient, and the clinically significant interaction persisted. The dose of everolimus could not be reduced further without sacrificing the clinical efficacy of everolimus.

In some embodiments, Applicant found that, in addition to dose reductions of everolimus, a dose reduction of CBD is required to safely and effectively treat seizures in TSC patients. In some embodiments, the reduced doses of CBD and everolimus that are effective when coadministered, are ineffective when CBD and everolimus are not coadministered (e.g. when the reduced dose of CBD is administered without everolimus or when everolimus is administered without CBD). In some embodiments, the reduced doses of CBD is less than the approved dose for TSC, but falls within levels approved for treating seizures associated with other forms of epilepsy (e.g., Lennox-Gastaut syndrome and Dravet syndrome).

For purposes of this disclosure, “reduced dose” may be referred to alternatively as a dose reduction, down dose, downward dose, or downward titration. For example, in some embodiments, the recommended dose of CBD for treating seizures with TSC is 25 mg/kg/day CBD. If the patient is coadminstered everolimus, the patient may downward titrate CBD to 20 mg/kg/day, 15 mg/kg/day, or 10 mg/kg/day. Similarly, a dose reduction, down dose or downward dose of CBD may required, desired, or considered to safely and effectively treat seizures associated with TSC.

In some embodiments, the disclosure provides for methods of treating seizures associated with TSC in a everolimus-näive patient, wherein the patient is currently treated with CBD, and the patient is in need of treatment with everolimus. As used herein, an “everolimus-näive patient” refers to a patient that is not currently being treated with everolimus. Rather, the everolimus-näive patient is currently treated with CBD to reduce seizures, and the patient is in need of everolimus. The term “everolimus näive patient” encompasses patients that may have been treated with everolimus previously, provided that they are not being treated with everolimus when receiving CBD treatment. In some embodiments, the method comprises monitoring the patient's blood plasma levels of everolimus, and reducing the dose of everolimus (e.g., by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) when everolimus trough plasma levels exceed 15 ng/mL, when Cmax(ss) is greater than or equal to about 46 ng/mL, about 40 ng/mL, about 35 ng/mL, about 30 ng/mL, about 25 ng/mL, or about 20 ng/mL (including all values and ranges therebetween), and/or when AUC(ss) is greater than or equal to about 530 h*ng/mL, about 500 h*ng/mL, about 450 h*ng/mL, about 400 h*ng/mL, about 350 h*ng/mL, about 300 h*ng/mL, about 250 h*ng/mL, or about 200 h*ng/mL (including all values and ranges therebetween).

Cannabidiol (CBD)

CBD has the following structure:

CBD is a cannabinoid, which may be produced synthetically, or extracted from Cannabis plants. In some embodiments, cannabidiol is extracted from Cannabis plants. Non-limiting examples of Cannabis plants include Cannabis sativa, Cannabis indica, or Cannabis ruderalis. In some embodiments, CBD is extracted from hybrid varieties of Cannabis. CBD may be extracted from Cannabis plants according to known methods in the art. Such extracts may be referred to interchangeably as a CBD extract, botanical extract, or botanical drug substance. Non-limiting extraction methods include sonication, heating under reflux, soxhlet extraction, solid-phase micro-extraction, supercritical-fluid extraction, pressurized-liquid extraction, microwave-assisted extraction, solid-phase extraction, and surfactant-mediated techniques. In some embodiments, the steps for extraction include, but are not limited, to pre-washing, drying of plant parts or freeze drying, and grinding to obtain homogenous extracted plant samples.

In some embodiments, cannabidiol is extracted using an alcohol-based extraction. In some embodiments, cannabidiol is extracted with ethanol. In some embodiments, cannabidiol is obtained using a supercritical carbon dioxide based extraction. Methods for extraction of CBD from Cannabis plants are described in the following patent documents which are incorporated by reference in their entirety herein: U.S. Publication No. 2019/0231833 A1, (published Aug. 1, 2019), International Publication No. 2019/020738 (published Jan. 31, 2019), International Publication No. 2004/016277 A1 (published Feb. 26, 2004), U.S. Publication No. 2019/0160393 A1 (published May 30, 2019), and International Publication No. 2004/026802 (published Jan. 4, 2004).

In some embodiments, cannabidiol is produced synthetically. As described herein, synthetic cannabidiol includes CBD analogs, CBD salts, modified CBD, and propyl cannabinoids (CBDv). Synthetic CBD has the same or similar therapeutic effects as naturally occurring CBD when administered to the subjects. Patent documents, such as U.S. Publication No. 2019/0031601 (published Jan. 31, 2019), U.S. Pat. No. 9,447,019 (issued Sep. 20, 2016), and U.S. Publication No. 2015/0320698 (published Nov. 12, 2015), which describe synthetic cannabinoids are incorporated by reference herein in their entirety.

Methods for cannabidiol synthesis are described in the following patent documents, which are incorporated by reference in their entirety herein: EP Publication No. 2578561 A1 (published Apr. 10, 2013), U.S. Publication No. 2017/0008868 A1 (published Aug. 28, 2018). In some embodiments, cannabidiol is produced in microorganisms. Methods for producing cannabidiol in microorganisms are described in U.S. Publication No. 2016/0010126 A1 (published Jan. 14, 2016) and International Publication No. 2017/139496 (published Aug. 17, 2016), which are incorporated by reference in their entireties, herein.

In some embodiments, CBD is present in a botanical extract. In such extracts, the CBD has a purity from about 95 w/w to about 100% w/w CBD. In some embodiments, the CBD has a purity of at least about 95% w/w, about 96% w/w, about 97% w/w, about 98% w/w, or about 99% w/w, including all values and ranges in between. In some embodiments, the CBD has a purity of at least about 98% w/w. In some embodiments, the CBD has a purity of at least 99% w/w. Accordingly, “CBD” as used herein refers to CBD having a purity ranging from about 95% to about 100%, e.g., at least about 98% w/w pure.

In some embodiments, the CBD extracts of the disclosure contain up to about 5% w/w of other cannabinoids and/or terpenes. In some embodiments, the CBD extracts of the disclosure contain up to about 2% w/w of other cannabinoids and/or terpenes. In some embodiments, the CBD extracts of the disclosure contain up to about 5% w/w, about 2% w/w, or up to about 1% of other cannabinoid, but is substantially free of terpenes. Indeed, the presence of these additional components may influence the pharmacological properties of the CBD administered according to the methods of the disclosure. Non-limiting examples of cannabinoids include cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol-C4 (CBD-C4), cannabidiol-C1 (CBD-C1), cannabielsoin-type-C1 (CBEI), cannabielsoin-type-C2 (CBEII), 6-OH-cannabidiol, 7-OH-cannabidiol, and tetrahydrocannabinolic acid (THCA). Non-limiting examples of terpenes include alpha-cedrene, alpha-humulene, alpha-pinene, alpha-terpinene, beta-myrcene, beta-pinene, borneol, camphene, camphor, caryophyllene oxide, cedrol, alpha-bisabolol, alpha-phellandrene, isopulegol, cis-nerolidol, 3-carene, fenchyl alcohol, hexahydrothymol, eucalyptol, isoborneol, farnesene, fenchone, gamma-terpinene, geraniol, geranyl acetate, humulene, guaiol, limonene, linalool, nerol, ocimene, alpha-phellandrene, pulegone, sabinene, sabinene hydrate, terpineol, terpinolene, trans-caryophyllene, β-caryophyllene, trans-nerolidol, and valencene.

In some embodiments, the CBD extracts of the disclosure contain up to about 1% w/w CBDV. In some embodiments, the CBD extracts of the disclosure contain about 0% w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w, about 0.25% w/w, about 0.30% w/w, about 0.35% w/w, about 0.40% w/w, about 0.45% w/w, about 0.50% w/w, about 0.55% w/w, about 0.60% w/w, about 0.65% w/w, about 0.70% w/w, about 0.75% w/w, about 0.80% w/w, about 0.85% w/w, about 0.90% w/w, about 0.95% w/w, or about 1.0% w/w CBDV, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain up to about 1 w/w THC. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.05 w/w, about 0.1% w/w, about 0.15 w/w, about 0.2% w/w, about 0.25 w/w, about 0.30% w/w, about 0.35 w/w, about 0.40% w/w, about 0.45 w/w, about 0.50 w/w, about 0.55% w/w, about 0.60% w/w, about 0.65 w/w, about 0.70% w/w, about 0.75 w/w, about 0.80% w/w, about 0.85 w/w, about 0.90% w/w, about 0.95 w/w, or about 1.0% w/w THC, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain up to about 0.5% w/w CBD-C4. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.05 w/w, about 0.1% w/w, about 0.15 w/w, about 0.2% w/w, about 0.25 w/w, about 0.30% w/w, about 0.35 w/w, about 0.40% w/w, about 0.45 w/w, or about 0.50 w/w CBD-C4.

In some embodiments, the CBD extracts of the disclosure contain up to about 0.15% w/w CBD-C1. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.005 w/w, about 0.01% w/w, about 0.015 w/w, about 0.02% w/w, about 0.025% w/w, about 0.030% w/w, about 0.035 w/w, about 0.040% w/w, about 0.045 w/w, about 0.050% w/w, about 0.055 w/w, about 0.060% w/w, about 0.065% w/w, about 0.070% w/w, about 0.075 w/w, about 0.080% w/w, about 0.085 w/w, about 0.090% w/w, about 0.095% w/w, about 0.10% w/w, or about 0.15% w/w CBD-C1, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain up to about 0.2% w/w CBEI. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.005 w/w, about 0.01% w/w, about 0.015 w/w, about 0.02% w/w, about 0.025% w/w, about 0.030% w/w, about 0.035 w/w, about 0.040% w/w, about 0.045 w/w, about 0.050% w/w, about 0.055 w/w, about 0.060% w/w, about 0.065% w/w, about 0.070% w/w, about 0.075 w/w, about 0.080% w/w, about 0.085 w/w, about 0.090% w/w, about 0.095% w/w, about 0.10% w/w, about 0.15 w/w, or about 0.20% w/w CBEI, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain up to about 0.2% w/w CBEII. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.005 w/w, about 0.01% w/w, about 0.015 w/w, about 0.02% w/w, about 0.025% w/w, about 0.030% w/w, about 0.035 w/w, about 0.040% w/w, about 0.045 w/w, about 0.050% w/w, about 0.055% w/w, about 0.060% w/w, about 0.065% w/w, about 0.070% w/w, about 0.075 w/w, about 0.080% w/w, about 0.085 w/w, about 0.090% w/w, about 0.095% w/w, about 0.10% w/w, about 0.15 w/w, or about 0.20% w/w CBEII, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain up to about 0.2% w/w of a combination of CBEI and CBEII. In some embodiments, the CBD extracts of the disclosure contain about 0 w/w, about 0.005 w/w, about 0.01% w/w, about 0.015 w/w, about 0.02% w/w, about 0.025 w/w, about 0.030% w/w, about 0.035 w/w, about 0.040% w/w, about 0.045 w/w, about 0.050 w/w, about 0.055 w/w, about 0.060% w/w, about 0.065% w/w, about 0.070% w/w, about 0.075% w/w, about 0.080% w/w, about 0.085% w/w, about 0.090% w/w, about 0.095 w/w, about 0.10% w/w, about 0.15 w/w, or about 0.20% w/w of a combination of CBEI and CBEII including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure contain less than about 0.1% w/w OH-CBD. In some embodiments, the CBD extracts of the disclosure contain about 0% w/w, about 0.005 w/w, about 0.01% w/w, about 0.015% w/w, about 0.02% w/w, about 0.025% w/w, about 0.030% w/w, about 0.035 w/w, about 0.040% w/w, about 0.045 w/w, about 0.050% w/w, about 0.055% w/w, about 0.060% w/w, about 0.065% w/w, about 0.070% w/w, about 0.075 w/w, about 0.080% w/w, about 0.085 w/w, about 0.090% w/w, about 0.095% w/w, or about 0.10% w/w OH-CBD, including all values and ranges in between.

In some embodiments, the CBD extracts of the disclosure comprise CBD, at purity of at least 95% w/w (e.g., 98% w/w, or 99% w/w), and CBDA, CBDV, THC and CBD-C4. In some embodiments, the CBDA is present in an amount of about 0.15% w/w or less, e.g., about 0.15% w/w, about 0.1% w/w, about 0.05% w/w, or about 0.01% w/w, inclusive of all values and ranges between these values. In some embodiments, CBDV is present in an amount of about 1.0% w/w or less, e.g., about 1.0% w/w or less, about 0.9% w/w, about 0.8% w/w, about 0.7% w/w, about 0.6% w/w, about 0.5% w/w, about 0.4% w/w, about 0.3% w/w, about 0.2% w/w, about 0.1% w/w, about 0.09% w/w, about 0.08% w/w, about 0.07% w/w, about 0.06% w/w, about 0.05% w/w, about 0.04% w/w, about 0.03% w/w, about 0.02% w/w, about 0.01% w/w, inclusive of all values and ranges between these values. In some embodiments, THC is present in an amount of about 0.15% w/w or less, e.g., about 0.15% w/w, about 0.1% w/w, about 0.05% w/w, or about 0.01% w/w, inclusive of all values and ranges between these values. In some embodiments, CBD-C4 is present in an amount of about 0.5% w/w or less, e.g., about 0.5% w/w, about 0.4% w/w, about 0.3% w/w, about 0.2% w/w, about 0.1% w/w, about 0.09% w/w, about 0.08% w/w, about 0.07% w/w, about 0.06% w/w, about 0.05% w/w, about 0.04% w/w, about 0.03% w/w, about 0.02% w/w, about 0.01% w/w, inclusive of all values and ranges between these values.

In some embodiments, CBD is administered at a dose from about 1 mg/kg/day to about 25 mg/kg/day. In some embodiments, CBD is administered at a dose from about 5 mg/kg/day to about 50 mg/kg/day. In some embodiments, CBD is administered at a dose of about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, or about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, or about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day, about 20 mg/kg/day, about 21 mg/kg/day, about 22 mg/kg/day, about 23 mg/kg/day, about 24 mg/kg/day, about 25 mg/kg/day, about 26 mg/kg/day, about 27 mg/kg/day, about 28 mg/kg/day, about 29 mg/kg/day, about 30 mg/kg/day, about 31 mg/kg/day, about 32 mg/kg/day, about 33 mg/kg/day, about 34 mg/kg/day, about 35 mg/kg/day, about 36 mg/kg/day, about 37 mg/kg/day, about 38 mg/kg/day, about 39 mg/kg/day, about 40 mg/kg/day, about 41 mg/kg/day, about 42 mg/kg/day, about 43 mg/kg/day, about 44 mg/kg/day, about 45 mg/kg/day, about 46 mg/kg/day, about 47 mg/kg/day, about 48 mg/kg/day, about 49 mg/kg/day, or about 50 mg/kg/day. In some embodiments, CBD is administered at a dose of about 5 mg/kg/day. In some embodiments, CBD is administered at a dose of about 10 mg/kg/day. In some embodiments, CBD is administered at a dose of about 15 mg/kg/day. In some embodiments, CBD is administered at a dose of about 20 mg/kg/day. In some embodiments, CBD is administered at a dose of about 25 mg/kg/day. In some embodiments, CBD is administered at a dose of about 50 mg/kg/day.

In some embodiments, CBD is administered at a dose of about 2.5 mg/kg to about 12.5 mg/kg twice daily. In some embodiments, CBD is administered at a dose from about 2.5 mg/kg to about 25 mg/kg twice daily. In some embodiments, CBD is administered at a dose of about 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, or about 11 mg/kg/day, about 12 mg/kg, about 12.5 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, or about 25 mg/kg. In some embodiments, CBD is administered at a dose of about 2.5 mg/kg twice daily. In some embodiments, CBD is administered at a dose of about 5 mg/kg twice daily. In some embodiments, CBD is administered at a dose of about 10 mg/kg twice daily. In some embodiments, CBD is administered at a dose of about 12.5 mg/kg twice daily.

As disclosed herein, in some embodiments, CBD is administered at a reduced dose when CBD is coadministered with everolimus. A “reduced dose” of CBD is less than the dose that an otherwise identical patient with TSC would receive if said patient were not coadministered everolimus. In other words, the reduced dose of CBD administered to a patient with TSC as a combination therapy is less than the dose of CBD administered to the same (or similar) TSC patient that is not coadministered everolimus.

In some embodiments, Applicant contemplates the dose of CBD administered to a TSC patient based on the approved dosing range of EPIDIOLEX® for treating seizures in patents with Lennox-Gastaut or Dravet Syndrome. In some embodiments, Applicant contemplates that the approved dosing range of CBD in the absence of everolimus for TSC will be 5 mg/kg/day to 25 mg/kg/day. In some embodiments, Applicant contemplates that patients will be treated with about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, or about 25 mg/kg/day in the absence of everolimus. In particular embodiments, Applicant contemplates that patients will be treated with about 10 mg/kg/day, about 15 mg/kg/day, or about 20 mg/kg/day, in the absence of everolimus.

Therapeutically effective doses will vary depending on the severity of the symptoms. As discussed herein, the methods of the present disclosure provide administering a reduce dose of CBD relative to the dose that would be therapeutically effective in the absence of everolimus. In some embodiments, the therapeutically effective dose of CBD for TSC will be 10 mg/kg/day. Accordingly, in embodiments, the reduced dose of CBD for such a patient is less than 10 mg/kg/day, e.g. 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, or 9 mg/kg/day.

In some embodiments, the therapeutically effective dose of CBD for TSC will be 15 mg/kg/day. Accordingly, in some embodiments, the reduced dose of CBD for such a patient is less than 15 mg/kg/day, e.g. 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, 9 mg/kg/day, 10 mg/kg/day, 11 mg/kg/day, 12 mg/kg/day, 13 mg/kg/day, or 14 mg/kg/day.

In some embodiments, the therapeutically effective dose of CBD for TSC will be 20 mg/kg/day. Accordingly, in some embodiments, the reduced dose of TSC for such a patient is less than 20 mg/kg/day, e.g. 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, or 9 mg/kg/day, 10 mg/kg/day, 11 mg/kg/day, 12 mg/kg/day, 13 mg/kg/day, 14 mg/kg/day, 15 mg/kg/day, 16 mg/kg/day, 17 mg/kg/day, 18 mg/kg/day, or 19 mg/kg/day.

In some embodiments, the therapeutically effective dose of CBD for TSC will be 25 mg/kg/day. Accordingly, in some embodiments, the reduced dose of CBD for such a patient is less than 25 mg/kg/day, e.g. 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, or 9 mg/kg/day, 10 mg/kg/day, 11 mg/kg/day, 12 mg/kg/day, 13 mg/kg/day, 14 mg/kg/day, 15 mg/kg/day, 16 mg/kg/day, 17 mg/kg/day, 18 mg/kg/day, 19 mg/kg/day, 20 mg/kg/day, 21 mg/kg/day, 22 mg/kg/day, 23 mg/kg/day, or 24 mg/kg/day.

In some embodiments, the reduced dose of CBD administered to a TSC patient in combination with everolimus is from about 10% to about 75% less than the dose of CBD administered to the same TSC patient in the absence of everolimus. In some embodiments, the reduced dose of CBD administered to a TSC patient in combination with everolimus is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% less than the dose of CBD administered to the same TSC patient in the absence of everolimus, including all values and ranges between these values.

In some embodiments, the reduced dose of CBD administered to a TSC patient in combination with everolimus is reduced by at least about 1 mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day, or about 20 mg/kg/day compared to the dose of CBD the patient would receive in the absence of everolimus, including all values and ranges between these values. In some embodiments, the reduced dose of CBD administered to a TSC patient in combination with everolimus is reduced by at least about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, or about 20 mg/kg compared to the dose of CBD the patient would receive in the absence of everolimus, including all values and ranges between these values.

In some embodiments, the reduced dose of CBD ranges from about 1 mg/kg/day to about 23 mg/kg/day. In some embodiments, the reduced dose of CBD is about 1 mg/kg/day, about 2 mg/kg/day, about 2.5 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day, about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, or about 11 mg/kg/day, about 12 mg/kg/day, about 12.5 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, or about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day, about 20 mg/kg/day, about 21 mg/kg/day, about 22 mg/kg/day, or about 23 mg/kg/day, including all values and ranges in between. In some embodiments, the reduced dose of CBD is about 5 mg/kg/day. In some embodiments, the reduced dose of CBD is about 10 mg/kg/day. In some embodiments, the reduced dose of CBD is about 15 mg/kg/day. In some embodiments, the reduced dose of CBD is about 20 mg/kg/day.

In some embodiments, coadminstration of CBD (at 25 mg/kg/day) with everolimus elevates the pharmacokinetics parameters (Cmax and AUC) of everolimus by 2.5 fold. In some embodiments, the dose of CBD is reduced such that the pharmacokinetics parameters (Cmax and AUC) of everolimus are increased by no more than 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0.

According to the current FDA label, CBD is administered at an starting dose of 5 mg/kg/day in patients with normal hepatic status or mild hepatic impairment. CBD causes dose-related elevations of liver transaminases (alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST)). A lower starting dose compared to maintenance dose (e.g. the lowest dose of drug that achieves efficacy and minimizes side effects) is important to adapt the liver to CBD, such that it can increase CBD metabolism and reduce the incidences of dose-related elevations of liver transaminases ALT and/or AST.

If the starting dose is tolerated and additional seizure reduction is required, the maintenance dose of CBD can be increased to a maximum dose of 25 mg/kg/day (in the absence of everolimus). Because CBD can cause elevation of liver transaminases, the serum concentration of these enzymes is monitored.

Due to the DDI with everolimus, in some embodiments, the starting dose of CBD is reduced from the currently approved starting dose of 5 mg/kg/day. In some embodiments, CBD (when coadministered with everolimus) will be administered at a starting dose from about 1 mg/kg/day to about 4 mg/kg/day (e.g., about 1 mg/kg/day, about 1.5 mg/kg/day, about 2 mg/kg/day, about 2.5 mg/kg/day, about 3 mg/kg/day, about 3.5 mg/kg/day, or about 4 mg/kg/day), and the dose of CBD will be increased up to maximum dose of e.g., about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, or 24 mg/kg/day.

In some embodiments, the starting dose of CBD may be increased for efficacy. In some embodiments, the dose of CBD may be increased in increments of about 0.5 mg/kg/day to about 5 mg/kg/day. In some embodiments the dose of CBD may be increased by about 0.5 mg/kg/day, about 1.0 mg/kg/day, about 1.5 mg/kg/day, about 2.0 mg/kg/day, about 2.5 mg/kg/day, about 3.0 mg/kg/day, about 3.5 mg/kg/day, about 4.0 mg/kg/day, about 4.5 mg/kg/day, or about 5.0 mg/kg/day.

In some embodiments, the dose of CBD may be increased every day, every other day, every third day, every fourth day, every fifth day, every sixth day, or every seventh day, every two weeks, every three weeks, every four weeks, every five weeks, every month, every two months, or every year. In some embodiments, the dose of CBD may be increased from the starting dose after one week. Dose increases may occur once per week thereafter.

In some embodiments, after administering between about 1 mg/kg/day and about 50 mg/kg/day of CBD (e.g. about 1.0 mg/kg/day, about 2.0 mg/kg/day, about 2.5 mg/kg/day, about 3.0 mg/kg/day, about 3.5 mg/kg/day, about 4.0 mg/kg/day, about 5.0 mg/kg/day, about 6.0 mg/kg/day, about 7.5 mg/kg/day, about 8 mg/kg/day, about 9 mg/kg/day, about 10 mg/kg/day, about 11 mg/kg/day, about 12 mg/kg/day, about 13 mg/kg/day, about 14 mg/kg/day, about 15 mg/kg/day, about 16 mg/kg/day, about 17 mg/kg/day, about 18 mg/kg/day, about 19 mg/kg/day, about 20 mg/kg/day, about 21 mg/kg/day, about 22 mg/kg/day, about 23 mg/kg/day, about 24 mg/kg/day, about 25 mg/kg/day, about 26 mg/kg/day, about 27 mg/kg/day, about 28 mg/kg/day, about 29 mg/kg/day, about 30 mg/kg/day, about 31 mg/kg/day, about 32 mg/kg/day, about 33 mg/kg/day, about 34 mg/kg/day, about 35 mg/kg/day, about 36 mg/kg/day, about 37 mg/kg/day, about 38 mg/kg/day, about 39 mg/kg/day, about 40 mg/kg/day, about 41 mg/kg/day, about 42 mg/kg/day, about 43 mg/kg/day, about 44 mg/kg/day, about 45 mg/kg/day, about 46 mg/kg/day, about 47 mg/kg/day, about 48 mg/kg/day, about 49 mg/kg/day, or about 50 mg/kg/day), the patient has an steady state area under the concentration time curve from time zero (t1) to five hours (t2) (AUC_(t1-t2)) between 25 ng*hr/mL and 4000 ng*hr/mL. In some embodiments, the AUC_(t1-t2) is about 25 ng*hr/mL, about 50 ng*hr/mL, about 75 ng*hr/mL, about 100 ng*hr/mL, about 125 ng*hr/mL, about 150 ng*hr/mL, about 175 ng*hr/mL, about 200 ng*hr/mL, about 225 ng*hr/mL, about 250 ng*hr/mL, about 275 ng*hr/mL, about 300 ng*hr/mL, about 325 ng*hr/mL, about 350 ng*hr/mL, about 375 ng*hr/mL, and about 400 ng*hr/mL, about 500 ng*hr/mL, about 600 ng*hr/mL, about 700 ng*hr/mL, about 800 ng*hr/mL, about 900 ng*hr/mL, about 1000 ng*hr/mL, about 1100 ng*hr/mL, about 1200 ng*hr/mL, about 1300 ng*hr/mL, about 1400 ng*hr/mL, about 1500 ng*hr/mL, about 1600 ng*hr/mL, about 1700 ng*hr/mL, about 1800 ng*hr/mL, about 1900 ng*hr/mL, about 2000 ng*hr/mL, about 2100 ng*hr/mL, about 2200 ng*hr/mL, about 2300 ng*hr/mL, about 2400 ng*hr/mL, about 2500 ng*hr/mL, about 2600 ng*hr/mL, about 2700 ng*hr/mL, about 2800 ng*hr/mL, about 2900 ng*hr/mL, about 3000 ng*hr/mL, about 3100 ng*hr/mL, about 3200 ng*hr/mL, about 3300 ng*hr/mL, about 3400 ng*hr/mL, about 3500 ng*hr/mL, about 3600 ng*hr/mL, about 3700 ng*hr/mL, about 3800 ng*hr/mL, about 3900 ng*hr/mL, about or about 4000 ng*hr/mL, including all ranges and values in between. In some embodiments, the AUC_(t1-t2) is between 80% and 125% of the aforementioned values.

In some embodiments, the AUC_(t1-t2) is reported as a geometric mean (% coefficient of variation). In some embodiments, after administration of everolimus and CBD, the patient has an AUC_(t1-t2) of CBD that is bioequivalent to 25 mg/kg/day of CBD—i.e., an AUC_(t1-t2) ranging from 80% to about 125% of 2520 (52.4%) ng*hr/mL. In some embodiments, after administration of everolimus and CBD, the patient has an AUC_(t1-t2) of CBD that is less than AUC_(t1-t2) of 50 mg/kg/day of CBD—i.e., less than 2730 (87.2%) ng*hr/mL (e.g., less than 2700 ng*hr/mL, 2600 ng*hr/mL, 2500 ng*hr/mL, 2400 ng*hr/mL, 2300 ng*hr/mL, 2200 ng*hr/mL, 2100 ng*hr/mL, 2000 ng*hr/mL, 1900 ng*hr/mL, 1800 ng*hr/mL, 1700 ng*hr/mL, 1600 ng*hr/mL, 1500 ng*hr/mL, 1400 ng*hr/mL, 1300 ng*hr/mL, 1200 ng*hr/mL, 1100 ng*hr/mL, or 1000 ng*hr/mL, etc).

In some embodiments, after administration of everolimus and CBD, the patient has a steady state AUC of CBD from time zero to the last detectable dose (t) (AUC_(0-t)) that is bioequivalent to 5 mg/kg/day of CBD—i.e., an AUC_(0-t) ranging from 80% to about 125% of 241 (101) ng*hr/mL. In some embodiments, after administration of everolimus and CBD, the patient has an AUC_(0-t) of CBD that is bioequivalent to 10 mg/kg/day of CBD—i.e., an AUC_(0-t) ranging from 80% to about 125% of 722 (79.9) ng*hr/mL. In some embodiments, after administration of everolimus and CBD, the patient has an AUC_(0-t) of CBD that is bioequivalent to 20 mg/kg/day of CBD—i.e., an AUC_(0-t) ranging from 80% to about 125% of 963 (93.4) ng*hr/mL.

Everolimus

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), a serine-threonine kinase, downstream of the PI3K/AKT pathway. Everolimus binds to the intracellular protein FKBP-12, resulting in an inhibitory complex formation with mTOR complex 1 (mTORC1) and inhibition of mTOR kinase activity. Everolimus reduces the activity of S6 ribosomal protein kinase (S6K1) and eukaryotic elongation factor 4E-binding protein (4E-BP1), downstream effectors of mTOR, which are involved in protein synthesis. S6K1 is a substrate of mTORC1 and phosphorylates the activation domain 1 of the estrogen receptor which results in ligand-independent activation of the receptor. In addition, everolimus inhibits the expression of hypoxia-inducible factor (e.g., HIF-1) and reduced the expression of vascular endothelial growth factor (VEGF). Inhibition of mTOR by everolimus has been shown to reduce cell proliferation, angiogenesis, and glucose uptake in in vitro and/or in vivo studies.

Constitutive activation of the PI3K/Akt/mTOR pathway can contribute to endocrine resistance in breast cancer. In vitro studies show that estrogen-dependent and HER2+ breast cancer cells are sensitive to the inhibitory effects of everolimus, and that combination treatment with everolimus and Akt, HER2, or aromatase inhibitors enhances the anti-tumor activity of everolimus in a synergistic manner.

Two regulators of mTORC1 signaling are the oncogene suppressors tuberin-sclerosis complexes 1 and 2 (TSC1, TSC2). Loss or inactivation of either TSC1 or TSC2 leads to activation of downstream signaling. In tuberous sclerosis complex, inactivating mutations in either the TSC1 or the TSC2 gene lead to hamartoma formation throughout the body.

Everolimus is approved by the FDA for treatment of the following conditions: treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC) in combination with exemestane after failure of treatment with letrozole or anstrozole; adults with progressive neuroendocrine tumors of pancreatic origin (PNET) that are unresectable, locally advanced or metastatic; adults with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib; adults with renal angiomyolipoma and TSC, not requiring immediate surgery; and, pediatric and adult patients with TSC who have subependymal giant cell astrocytoma (SEGA) that requires therapeutic intervention but cannot be curatively resected; the prophylaxis of organ rejection in adult patients, including kidney transplant in combination with basiliximab, cyclosporine (reduced doses) and corticosteroids and liver transplant in combination with tacrolimus (reduced doses) and corticosteroids.

Everolimus has the following structure:

The recommended dosage of everolimus for TSC-associated renal angiomyolipoma is 10 mg orally once daily. The recommended dosage of everolimus for TSC-associated subependymal gain cell astrocytoma (SEGA) is 4.5 mg/² orally once daily. The recommended dosage of everolimus for TSC-associated partial-onset seizures is 5 mg/² orally once daily

In some embodiments, everolimus is administered at a dose ranging from about 2 mg to about 10 mg. In some embodiments, about 2 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 6.5 mg, about 7.0 mg, about 7.5 mg, about 8.0 mg, about 8.5 mg, about 9.0 mg, about 9.5 mg, or about 10.0 mg of everolimus is administered. In some embodiments, the dose of everolimus is 2.5 mg of everolimus. In some embodiments, the dose of everolimus is 5 mg of everolimus. In some embodiments, the dose of everolimus is 7.5 mg of everolimus. In some embodiments, the dose of everolimus is 10 mg of everolimus. In some embodiments, if the patient's body surface area (BSA) ranges from 0.5 m² to 1.2 m², the dose of everolimus is 2.5 mg once daily. In some embodiments, if the patient's BSA ranges from 1.3 m² to 2.1 m², the dose of everolimus is 5 mg once daily. In some embodiments, if the patient's BSA is greater than or equal to 2.2 m², the dose of everolimus is 7.5 mg once daily.

In some embodiments, everolimus is administered at a dose ranging from about 2.5 mg/m² to about 5.5 mg/m², e.g., about 2.5 mg/m², about 3.0 mg/m², about 3.5 mg/m², about 4.0 mg/m², about 4.5 mg/m², about 5.0 mg/m², or about 5.5 mg/m², inclusive of all values and ranges between these values. In some embodiments, the dose of everolimus is 5.0 mg/m² of everolimus. In some embodiments, the dose of everolimus is 4.5 mg/m² of everolimus. In some embodiments, the dose of everolimus is 2.5 mg/m² of everolimus.

In some embodiments, everolimus is administered in an amount sufficient to attain trough concentrations of 5 ng/mL to 15 ng/mL. When trough levels exceed 15 ng/mL are not tolerable, and present a risk of adverse events, such as gastrointestinal disorders (vomiting, diarrhea, constipation), hyperlipidemia, respiratory tract infection, nasopharyngitis, pyrexia, interstitial pneumonitis, edema, mouth ulcers, impaired wound healing, hematotoxicity, stomatitis, renal failure, and rash.

In some embodiments, everolimus is administered once daily.

In some embodiments, everolimus is administered every day, every other day, every third day, every fourth day, every fifth day, every sixth day, every week, every 2 weeks, every month, or every year. In some embodiments, everolimus is administered every day. In some embodiments, everolimus is administered every other day.

In some embodiments, a starting dose of about 4.5 mg/m² of everolimus is administered, and the dose of everolimus is adjusted to obtain trough concentrations of about 5 ng/mL to about 15 ng/mL.

In some embodiments, a starting dose of about 5.0 mg/m² of everolimus is administered, and the dose of everolimus is adjusted to obtain trough concentrations of about 5 ng/mL to about 15 ng/mL. In some embodiments, the dose is adjusted to about 5.5 mg/m², about 6 mg/m², about 6.5 mg/m², about 7 mg/m², about 7.5 mg/m², about 8 mg/m², about 8.5 mg/m², 9 mg/m², about 9.5 mg/m², or to about 10 mg/m², including all values and ranges between these values.

In some embodiments, the dose of everolimus is increased from the starting dose. In some embodiments, the dose of everolimus is decreased from the starting dose. In some embodiments, the dose of everolimus administered is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including all values and ranges therebetween. In some embodiments, the dose reduction of everolimus

Everolimus is metabolized by CYP3A4, CYP3A5, and CYP2C8. Following oral administration of everolimus, everolimus is the main circulating component in the blood. Six main metabolites of everolimus have been detected in human blood, including three monohydroxylated metabolites, two hydrolytic ring-opened products, and a phosphatidylcholine conjugate of everolimus.

Everolimus is a substrate of CYP3A4 and P-gp. The FDA label for everolimus suggests avoiding the concomitant use of strong P-gp and strong CYP3A4 inhibitors and reducing the dose of everolimus with a P-gp and moderate CYP3A4 inhibitor (Table 1 and Table 2).

TABLE 1 Recommended Dosage Modifications for Concurrent Use of Everolimus with a P-gp and moderate CYP3A4 inhibitor Indication Dose Modification for Everolimus Breast Cancer, NET, RCC, Reduce dose to 2.5 mg once daily. and TSC-Associated Renal May increase dose to 5 mg once daily Angiomyolipoma if tolerated. Resume dose administered prior to inhibitor initiation, once the inhibitor is discontinued for 3 days. TSC-Associated SEGA and Reduce the daily dose by 50%. TSC-Associated Partial- Change to every other day dosing if Onset Seizures the reduced dose is lower than the lowest available strength. Resume dose administered prior to inhibitor initiation, once the inhibitor is discontinued for 3 days.

TABLE 2 Recommended Dosage Modifications for Concurrent Use of Everolimus with a P-gp and strong CYP3A4 inducer Indication Dose Modification for Everolimus Breast Cancer, NET, RCC, Avoid coadministration where and TSC-Associated Renal alternatives exist. Angiomyolipoma If coadministration cannot be avoided, double the daily dose using increments of 5 mg or less. Multiple increments may be required. Resume dose administered prior to inducer initiation, once the inducer is discontinued for 5 days. TSC-Associated SEGA and Double the daily dose using TSC- Associated Partial- increments of 5 mg or less. Multiple Onset Seizures increments may be required. Addition of another strong CYP3A4 inducer in a patient already receiving treatment with a strong CYP3A4 inducer may not require additional dosage Assess trough concentrations when initiating and discontinuing the inducer Resume the dose administered before starting any inducer, once all inducers are discontinued for 5 days.

After administration of everolimus in patients with advanced solid tumors, peak everolimus concentrations are reached 1 to 2 hours after administration of oral doses ranging from 5 mg to 70 mg. Following single doses, Cmax is dose proportional with daily dosing between 5 mg and 10 mg. With single doses of 20 mg and higher, the increase in Cmax is less than dose-proportional; however, AUC shows dose-proportionality over the 5 mg to 70 mg dose range. Steady-state was achieved within 2 weeks following once-daily dosing. In patients with TSC-associated SEGA, everolimus Cmin was approximately dose-proportional within the dose range from 1.35 mg/m² to 14.4 mg/m².

Everolimus area under the curve (AUC) is linear in the dose range from 2.5 mg to 70 mg.

In some embodiments, after administering between about 1 mg and about 20 mg (e.g., 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.5 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg) of everolimus, the patient has a maximum observed plasma drug concentration (C_(maxSS)) between 1 ng/mL and 200 ng/mL. In some embodiments, the C_(maxSS) is about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 21.6 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 mg/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 105 ng/mL, about 110 ng/mL, about 115 ng/mL, about 120 ng/mL, about 125 ng/mL, about 130 ng/mL, about 135 ng/mL, about 140 ng/mL, about 145 ng/mL, about 150 ng/mL, about 155 ng/mL, about 160 ng/mL, about 165 ng/mL, about 170 ng/mL, about 175 ng/mL, about 180 ng/mL, about 185 ng/mL, about 190 ng/mL, about 195 ng/mL, or about 200 ng/mL, including all ranges and values in between. In some embodiments, the C_(maxSS) is between 80% and 125% of any of the aforementioned values or ranges between the aforementioned values.

In some embodiments, the C_(maxSS) is reported as a geometric mean±Standard Deviation (SD). In some embodiments, after administration of everolimus and CBD, the patient has a C_(maxSS) of everolimus that is bioequivalent to 2.5 mg of everolimus—i.e., a C_(maxSS) ranging from 80% to about 125% of 62.1±13.1 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(maxSS) of everolimus that is bioequivalent to 5 mg of everolimus—i.e., a C_(maxSS) ranging from 80% to about 125% of 114±36 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(maxSS) of everolimus that is bioequivalent to 10 mg of everolimus—i.e., a C_(maxSS) ranging from 80% to about 125% of 139 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(maxSS) of everolimus that within the range of the C_(maxSS) of 2.5 mg and C_(maxSS) the of 10 mg.

In some embodiments, after administering between about 1 mg and about 20 mg (e.g., 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.5 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg) of everolimus, the patient has a minimum observed steady state plasma drug concentration (C_(minSS)) of everolimus between 1 ng/mL and 100 ng/mL. In some embodiments, the C_(minSS) is about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 21.6 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 mg/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL, including all ranges and values in between. In some embodiments, the C_(minSS) is between 80% and 125% of any of the aforementioned values or ranges between the aforementioned values.

In some embodiments, the C_(minSS) is reported as a geometric mean±Standard Deviation (SD). In some embodiments, after administration of everolimus and CBD, the patient has a C_(minSS) of everolimus that is bioequivalent to 2.5 mg of everolimus—i.e., a C_(minSS) ranging from 80% to about 125% of 13.6±9.0 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(minSS) of everolimus that is bioequivalent to 5 mg of everolimus—i.e., a C_(minSS) ranging from 80% to about 125% of 17.4±4.5 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(minSS) of everolimus that is bioequivalent to 10 mg of everolimus—i.e., a C_(minSS) ranging from 80% to about 125% of 16.5 ng/mL. In some embodiments, after administration of everolimus and CBD, the patient has a C_(maxSS) that within the range of the C_(minSS) of 2.5 mg and C_(minSS) the of 10 mg.

In some embodiments, after administering between about 1.0 mg and about 20 mg of everolimus, the patient has a time to reach C_(max) (t_(max)) between 0.1 hours and 2 hours. In some embodiments, the t_(max) is about 0.1 hours, about 0.2 hours, about 0.3 hours, about 0.4 hours, about 0.5 hours, about 0.6 hours, about 0.7 hours, about 0.8 hours, about 0.9 hours, about 1.0 hours, about 1.1 hours, about 1.2 hours, about 1.3 hours, about 1.4 hours, about 1.5 hours, about 1.6 hours, about 1.7 hours, about 1.8 hours, about 1.9 hours, or about 2.0 hours. In some embodiments, the t_(max) is between about 80% and about 125% of any of the aforementioned values or ranges between the aforementioned values.

In some embodiments, the t_(max) is reported as a median (range). In some embodiments, the patient has a t_(max) ranging from 80% to about 125% of 1.0 hour (1.0 hour-1.5 hours) after administration of 2.5 mg of everolimus. In some embodiments, the patient has a t_(max) ranging from 80% to about 125% of 0.8 hour (0.8 hours-1.5 hours) after administration of 5 mg of everolimus. In some embodiments, the patient has a t_(max) ranging from 80% to about 125% of 1.0 hours after administration of 10 mg of everolimus.

In some embodiments, after administering between about 1.0 mg and about 20 mg of everolimus (e.g. 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.5 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg), the patient has an area under the concentration time curve from zero hours to twenty four hours (AUC₂₄) between 100 ng*hr/mL and 2000 ng*hr/mL. In some embodiments, the AUC₂₄ is about 25 ng*hr/mL, about 50 ng*hr/mL, about 75 ng*hr/mL, about 100 ng*hr/mL, about 125 ng*hr/mL, about 150 ng*hr/mL, about 175 ng*hr/mL, about 200 ng*hr/mL, about 225 ng*hr/mL, about 250 ng*hr/mL, about 275 ng*hr/mL, about 300 ng*hr/mL, about 325 ng*hr/mL, about 350 ng*hr/mL, about 375 ng*hr/mL, and about 400 ng*hr/mL, about 500 ng*hr/mL, about 600 ng*hr/mL, about 700 ng*hr/mL, about 800 ng*hr/mL, about 900 ng*hr/mL, about 1000 ng*hr/mL, about 1100 ng*hr/mL, about 1200 ng*hr/mL, about 1300 ng*hr/mL, about 1400 ng*hr/mL, about 1500 ng*hr/mL, about 1600 ng*hr/mL, about 1700 ng*hr/mL, about 1800 ng*hr/mL, about 1900 ng*hr/mL, or about 2000 ng*hr/mL including all ranges and values in between. In some embodiments, the AUC₂₄ is between 80% and 125% of the aforementioned values.

In some embodiments, the AUC₂₄ is reported as a geometric mean±Standard Deviation (SD). In some embodiments, after administration of everolimus and CBD, the patient has an AUC₂₄ that is bioequivalent to 2.5 mg of everolimus—i.e., an AUC₂₄ ranging from 80% to about 125% of 570±195 ng*hr/mL. In some embodiments, after administration of everolimus and CBD, the patient has an AUC₂₄ that is bioequivalent to 5 mg of everolimus—i.e., an AUC₂₄ ranging from 80% to about 125% of 716±123 ng*hr/mL. In some embodiments, after administration of everolimus and CBD, the patient has an AUC₂₄ that is bioequivalent to 10 mg of everolimus—i.e., an AUC₂₄ ranging from 80% to about 125% of 1470 ng*hr/mL.

In some embodiments, the methods of disclosure provide for dose reductions to achieve everolimus blood plasma trough concentrations ranging from 5-15 ng/mL, e.g., about 5, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL or 15 ng/mL, inclusive of all values and ranges between these values.

In some embodiments, the methods of the disclosure provide for dose reductions to achieve maximum blood plasma concentrations of everolimus less than 46 mg/mL, e.g., about 45 ng/mL, about 44 ng/mL, about 43 ng/mL, about 42 ng/mL, about 41 ng/mL, about 40 ng/mL, about 39 ng/mL, about 38 ng/mL, about 37 ng/mL, about 36 ng/mL, 35 ng/mL, about 34 ng/mL, about 33 ng/mL, about 32 ng/mL, about 31 ng/mL, about 30 ng/mL, about 29 ng/mL, about 28 ng/mL, about 27 ng/mL, about 26 ng/mL, 25 ng/mL, about 24 ng/mL, about 23 ng/mL, about 22 ng/mL, about 21 ng/mL, about 20 ng/mL, about 19 ng/mL, about 18 ng/mL, about 17 ng/mL, about 16 ng/mL, 15 ng/mL, inclusive of all values and ranges between these values.

In some embodiments, the methods of the disclosure provide for dose reductions to achieve an AUC_(0-last) of less than 500 n*ng/mL, e.g., about 490 n*ng/mL, about 480 n*ng/mL, about 460 n*ng/mL, about 450 n*ng/mL, about 440 n*ng/mL, about 430 n*ng/mL, about 420 n*ng/mL, about 410 n*ng/mL, about 400 n*ng/mL, about 390 n*ng/mL, about 380 n*ng/mL, about 360 n*ng/mL, about 350 n*ng/mL, about 340 n*ng/mL, about 330 n*ng/mL, about 320 n*ng/mL, about 310 n*ng/mL, about 200 n*ng/mL, about 190 n*ng/mL, about 180 n*ng/mL, about 160 n*ng/mL, about 150 n*ng/mL, about 140 n*ng/mL, about 130 n*ng/mL, about 120 n*ng/mL, about 110 n*ng/mL, or about 100 n*ng/mL, inclusive of all values and ranges therebetween.

In some embodiments, the methods of the disclosure provide for dose reductions to achieve an AUC_(0-inf) of less than 530 n*ng/mL, e.g., about 520 n*ng/mL, about 510 n*ng/mL, about 500 n*ng/mL, about 490 n*ng/mL, about 480 n*ng/mL, about 460 n*ng/mL, about 450 n*ng/mL, about 440 n*ng/mL, about 430 n*ng/mL, about 420 n*ng/mL, about 410 n*ng/mL, about 400 n*ng/mL, about 390 n*ng/mL, about 380 n*ng/mL, about 360 n*ng/mL, about 350 n*ng/mL, about 340 n*ng/mL, about 330 n*ng/mL, about 320 n*ng/mL, about 310 n*ng/mL, about 200 n*ng/mL, about 190 n*ng/mL, about 180 n*ng/mL, about 160 n*ng/mL, about 150 n*ng/mL, about 140 n*ng/mL, about 130 n*ng/mL, about 120 n*ng/mL, about 110 n*ng/mL, or about 100 n*ng/mL, inclusive of all values and ranges therebetween.

Methods of Evaluating CBD/Everolimus Efficacy

The therapeutically effective dose of CBD may be determined in clinical trials. For example, in some embodiments, dose-escalation may be to determine the therapeutically effective dose of CBD. Dose-escalation studies are describe, for example, in NCT02544750. The efficacy of a particular dose may be evaluated by determining the incidence of adverse events and the efficacy of the particular dose. Non-limiting examples of measures to determine efficacy include change in seizure frequency, number of treatment responders, number of patients with worsening, no change, or improvements in seizure frequency, change in composite focal seizure score, change in number of seizure-free days, change in the number of seizures by subtype, change in overall condition as assessed by the participant/caregiver, change in overall condition as assessed by the physician, change in the Vineland-II score, change in Wechscler score by subtest, change in Aberrant Behavior Checklist score, change in social communication questionnaire score, change in quality of life score, change in serum IGF-1 levels, the number of participants with changes in Tanner stage, an incidence of suicidality. Clinical outcomes are generally evaluated based on changes from baseline. As used herein, “baseline” refers to a 4 week period prior to starting treatment with CBD during which clinical outcomes are measured.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring changes in seizure frequency from baseline. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in seizure frequency of at least 5%. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in seizure frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits at least about a 50% decrease in seizure frequency.

In some embodiments, after treatment according to the methods of the disclosure, a patient exhibits a decrease in the number of seizures from baseline selected from the group consisting of absence, myoclonic, focal sensory, infantile, and epileptic spasms. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in myoclonic seizure frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in focal sensory seizure frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in absence seizure frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in infantile spasm frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between. In some embodiments, a patient that is treated according to the methods of the disclosure exhibits a decrease in epileptic spasm frequency of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the number of treatment responders. In some embodiments, at least about 20% of patients exhibit improved symptoms of TSC after treatment according to the methods of the disclosure. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, including all values and ranges in between, exhibit improved symptoms of TSC after treatment according to the methods of the disclosure. In some embodiments, the percent change is of seizures is used to categorize the efficacy of treatments. Patients are categorized into the following groups:

(a) >25% increase in TSC seizure frequency=worsening

(b) −25%-25% change in TSC seizure frequency=no change in TSC

(c) 25-50% decrease in TSC seizure frequency=improvement in TSC

(d) 50-75% decrease in TSC seizure frequency=improvement in TSC

(e) >75% decrease in TSC seizure frequency=improvement in TSC

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the number of patients with worsening, no change, or improvements in seizure frequency. In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the number of patients with improvements in seizure frequency compared to before said treating. In some embodiments, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% of patients exhibit improvements seizure frequency after treatment according to the methods of the disclosure compared to before said treating.

In some embodiments, patients treated according to the methods of the disclosure exhibit a reduction in seizure frequency compared to before said treating. In some embodiments, patients exhibit an about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% reduction in seizure frequency compared to before said treating. In some embodiments, patients exhibit a ≥50% reduction in TSC-associated seizure frequency.

In some embodiments, patients treated according to the methods of the disclosure exhibit a reduction in the number of episodes of convulsive and non-convulsive status epilepticus. In some embodiments, after treating according to the methods of the disclosure, the number of episodes of convulsive and non-convulsive status epilepticus decreased by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between, compared to baseline. In some embodiments, patients treated according to the methods of the disclosure exhibit a reduction in the number of episodes of convulsive status epilepticus. In some embodiments, after treating according to the methods of the disclosure, the number of episodes of convulsive status epilepticus decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between. In some embodiments, patients treated according to the methods of the disclosure exhibit a reduction in the number of episodes of non-convulsive status epilepticus. In some embodiments, after treating according to the methods of the disclosure, the number of episodes of non-convulsive status epilepticus decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between, compared to baseline.

In some embodiments, efficacy of the treatment according to the methods of the disclosure is determined by evaluating a patient's use of rescue medications. Non-limiting examples of rescue medications include benzodiazepines including diazepam, lorazepam, and midazolam. In some embodiments, after treating according to the methods of the disclosure, a patient's use of rescue medications decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in composite focal seizure score. In some embodiments, the composite focal seizure is calculated as the sum of: (1×Number of focal motor seizures without impairment of consciousness or awareness)+(2×Number of focal seizures with impairment of consciousness or awareness)+(3×number of focal seizures evolving to bilateral convulsive seizures). In some embodiments, after treating according to the methods of the disclosure, the composite focal seizure score decreases compared to prior to said treatment. In some embodiments, the composite focal seizure score decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, or about 100%, including all values and ranges in between.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the number of seizure-free days. In some embodiments, after treating according to the methods of the disclosure, the number of seizure-free days increases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, or more, including all values and ranges in between.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the number of seizures by subtype. Non-limiting examples of seizure subtypes experienced by TSC patients include infantile spasm, focal, partial, tonic-clonic, atonic, myoclonic, and absence. In some embodiments, treating according to the methods of the disclosure leads to a decrease in one or more seizure subtype. In some embodiments, treating according to the methods of the disclosure leads to an about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or higher reduction in the frequency of one or more seizure subtypes.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the changes in the Vineland Adaptive Behavior Scales (VABS). The Vineland Adaptive Behavior Scales (VABS), third edition, is a standardized measure of adaptive behavior used to evaluate the personal and social skills of an individual from birth through adulthood. Individuals can be evaluated on the VABS scale by either teachers or caregivers. According to the VABS, an individual is assigned a VABS adaptive behavior composite score, which measures an individual's functioning compare to others of his or her age. An individual is also assigned domain scores in communication, daily living skills, socialization, and motor skills to assess an individual's adaptive behavior strengths and weaknesses. An individual receives a score from 20 to 140 on each of the domain scores and the VABS adaptive behavior composite score. A score from 20 to 70 represents low adaptive level. A score from 71 to 85 represents moderately low adaptive level. A score from 86 to 114 represents moderately adequate adaptive level. A score from 115 to 129 represents moderately high adaptive level. A score from 130 to 140 represents high adaptive level.

In some embodiments, after said treatment the patient experiences a reduction of symptoms associated with TSC that is characterized by at least a one point increase in the VABS adaptive behavior composite score compared to prior to the treatment. In some embodiments, the increase in the VABS adaptive behavior score is about 1 point, about 2 points, about 3 points, about 4 points, about 5 points, about 6 points, about 7 points, about 8 points, about 9 points, about 10 points, about 11 points, about 12 points, about 13 points, about 14 points, about 15 points, about 16 points, about 17 points, about 18 points, about 19 points, about 20 points, about 21 points, about 22 points, about 23 points, about 24 points, about 25 points, about 26 points, about 27 points, about 28 points, about 29 points, about 30 points, about 31 points, about 32 points, about 33 points, about 34 points, about 35 points, about 36 points, about 37 points, about 38 points, about 39 points, about 40 points, about 41 points, about 42 points, about 43 points, about 44 points, about 45 points, about 46 points, about 47 points, about 48 points, about 49 points, about 50 points, about 51 points, about 52 points, about 53 points, about 54 points, about 55 points, about 56 points, about 57 points, about 58 points, about 59 points, about 60 points, about 61 points, about 62 points, about 63 points, about 64 points, about 65 points, about 66 points, about 67 points, about 68 points, about 69 points, or about 70 points compared to prior to said treating.

In some embodiments, the increase in VABS adaptive behavior composite score is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% compared to prior to said treating.

In some embodiments, after said treatment the patient experiences an increase in sociability that is characterized by an increase in the VABS adaptive behavior socialization domain score compared to prior to the treatment. In some embodiments, the increase in the VABS adaptive behavior socialization domain score is about 1 point, about 2 points, about 3 points, about 4 points, about 5 points, about 6 points, about 7 points, about 8 points, about 9 points, about 10 points, about 11 points, about 12 points, about 13 points, about 14 points, about 15 points, about 16 points, about 17 points, about 18 points, about 19 points, about 20 points, about 21 points, about 22 points, about 23 points, about 24 points, about 25 points, about 26 points, about 27 points, about 28 points, about 29 points, about 30 points, about 31 points, about 32 points, about 33 points, about 34 points, about 35 points, about 36 points, about 37 points, about 38 points, about 39 points, about 40 points, about 41 points, about 42 points, about 43 points, about 44 points, about 45 points, about 46 points, about 47 points, about 48 points, about 49 points, about 50 points, about 51 points, about 52 points, about 53 points, about 54 points, about 55 points, about 56 points, about 57 points, about 58 points, about 59 points, about 60 points, about 61 points, about 62 points, about 63 points, about 64 points, about 65 points, about 66 points, about 67 points, about 68 points, about 69 points, or about 70 points compared to prior to said treating.

In some embodiments, the increase in VABS adaptive behavior socialization domain score is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%, compared to prior to said treating. In some embodiments, the patient experiences an at least 10% improvement on the socialization domain score of VABS after treatment. In some embodiments, the patient experiences an at least 35% improvement on the socialization domain score of VABS after treatment.

In some embodiments, after said treatment the patient experiences an improvement in communication that is characterized by an increase in the VABS adaptive behavior communication domain score compared to prior to the treatment. In some embodiments, the increase in the VABS adaptive behavior communication domain is about 1 point, about 2 points, about 3 points, about 4 points, about 5 points, about 6 points, about 7 points, about 8 points, about 9 points, about 10 points, about 11 points, about 12 points, about 13 points, about 14 points, about 15 points, about 16 points, about 17 points, about 18 points, about 19 points, about 20 points, about 21 points, about 22 points, about 23 points, about 24 points, about 25 points, about 26 points, about 27 points, about 28 points, about 29 points, about 30 points, about 31 points, about 32 points, about 33 points, about 34 points, about 35 points, about 36 points, about 37 points, about 38 points, about 39 points, about 40 points, about 41 points, about 42 points, about 43 points, about 44 points, about 45 points, about 46 points, about 47 points, about 48 points, about 49 points, about 50 points, about 51 points, about 52 points, about 53 points, about 54 points, about 55 points, about 56 points, about 57 points, about 58 points, about 59 points, about 60 points, about 61 points, about 62 points, about 63 points, about 64 points, about 65 points, about 66 points, about 67 points, about 68 points, about 69 points, or about 70 points, compared to prior to the treatment.

In some embodiments, the increase in VABS adaptive behavior communication domain score is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% compared to prior to said treating.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the Wechscler Adult Intelligence Scale, fourth edition (WAIS-IV) score by subtest. The Wechsler Adult Intelligence Scale (WAIS-IV) is an IQ test designed to measure intelligence and cognitive ability in adults and older adolescents. In some embodiments, after treating according to the methods of the disclosure, the Weschsler Intelligence Scale for Children shows an improvement by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% in an adult or older adolescent's intellectual ability. In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the Weschsler Preschool and Primary Scale of Intelligence. In some embodiments, the composite score after treating according to the methods of the disclosure increases. In some embodiments, the composite score after treating according to the methods of the disclosure increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between. In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the Weschsler Intelligence Scale for Children. In some embodiments, after treating according to the methods of the disclosure, the Weschsler Intelligence Scale for Children shows an improvement by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% in a child's intellectual ability.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in the aberrant behavior checklist score. The aberrant behavior checklist (ABC) is a behavior rating scale that is utilized to rate individuals in five subscales: (1) irritability, agitation, and crying, (2) lethargy, social withdrawal, (3) stereotypic behavior, (4) hyperactivity and noncompliance, and (5) inappropriate speech. Individuals can be evaluated on the ABC by any adult that knows the individual well. The ABC contains a 58-item questionnaire that is utilized to assess the five-subscales. Each item on the 58-item questionnaire is rated from 0 to 3. A score of 0 means an absence in symptom. A score of 3 means an individual experiences the symptom with high severity. Items within each subscale are added to obtain a subscale score. Possible subscale scores on the ABC range from 0 to 48 with higher subscores indicating behavioral impairment. In some embodiments, after treating according to the methods of the disclosure, the patient experiences a reduction of symptoms associated with TSC that is characterized by at least a one point decrease in the ABC irritability agitation and crying (ABC-I) subscore. In some embodiments, after said treatment the patient experiences a reduction in irritability that is characterized by a decrease in the ABC-I communication domain score compared to prior to the treatment.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in social communication questionnaire score. TSC patients often have autism spectrum disorders (ASD). The social communication questionnaire is a tool used by physicians to screen patients with ASD. An individual's caregiver rates the verbal individual on a score of 0 to 39 or the non-verbal individual on a scale of 0 to 33. An individual that has ASD is characterized by a social communication questionnaire that is above 15. In some embodiments, after said treatment the patient experiences a reduction of symptoms associated with ASD that is characterized by an at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% decrease in the social communication questionnaire scale compared to prior to said treatment.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring changes in a patient's quality of life. In some embodiments, a change in the quality of life in childhood epilepsy (QOLCE) questionnaire is utilized to determine efficacy of the methods of the disclosure. The maximum QOLCE score is 100. Higher scores are correlated with improved quality of life. In some embodiments, the QOLCE score increases after treatment according to the methods of the disclosure by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between. In some embodiments, efficacy of treatment according to the methods of the disclosure is evaluated using the Quality of Life in Epilepsy (QOLIE-31-P) score. In some embodiments, after treatment according to the methods of the disclosure, the QOLIE-31-P score decreases compared to prior to said treatment. In some embodiments, the QOLIE-31-P decreases by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between.

In some embodiments, a patient's quality of life is assessed using the physician global impression of change (PGIC) score. In some embodiments, patients are assigned a score of 1 to 7, where 1 means that a patient is very much improved, and 7 means that a patient's condition has worsened. In some embodiments, after treating according to the methods of the disclosure, the PGIC decreases by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, including all values and ranges in between.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the change in serum insulin-like growth factor-1 (IGF-1) levels. IGF-1 is released after activation of the mTOR pathway. In some embodiments, after treating according to the methods of the disclosure, IGF-1 is reduced compared to prior to said treatment by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by evaluating a patient's risk for suicide using the Columbia-Suicide Severity Rating Scale (C-SSRS; 19+Years or C-SSRS Children's (6-18 years) score.

In some embodiments, efficacy of treatment according to the methods of the disclosure is determined by measuring the number of inpatient hospitalizations due to epilepsy. In some embodiments, after treating according to the methods of the disclosure the number of inpatient hospitalizations for a patient decreases. In some embodiments, the number of inpatient hospitalizations decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

Patient Populations

In some embodiments, the methods of this disclosure are utilized to treat patients with TSC.

More than half of patients with TSC also develop autism spectrum disorders (ASDs). In some embodiments, the methods of the disclosure are utilized to treat TSC patients that have ASDs.

In some embodiments, the methods of the disclosure are utilized to treat patients from age 0-80. In some embodiments, the methods of the present disclosure are utilized to treat patients from about age 0 to about age 22. In some embodiments, the patients are about 0 years old, about 1 year old, about 2 years old, about 3 years old, about 4 years old, about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old, about 16 years old, about 17 years old, about 18 years old, about 19 years old, about 20 years old, about 21 years old, or about 22 years old.

In some embodiments, the patients are administered CBD and/or everolimus based on the patient's body surface area (BSA). In some embodiments, patients exhibit a BSA between about 0.25 and 2.5. In some embodiments, patients have a BSA of about 0.25, or about 0.30, or about 0.35, or about 0.40, or about 0.45, or about 0.50, or about 0.55, or about 0.60, or about 0.65, or about 0.70, or about 0.75, or about 0.80, or about 0.85, or about 0.90, or about 0.95, or about 1.0, or about 1.1, or about 1.2, or about 1.3, or about 1.4, or about 1.5, or about 1.6, or about 1.7, or about 1.8, or about 1.9, or about 2.0, or about 2.1, or about 2.2, or about 2.3, or about 2.3, or about 2.4, or about 2.5, or more, including all values and ranges in between. In some embodiments, a patient's body surface area is calculated according to the following formula: BSA=weight (kg)^(0.425)×height (cm)^(0.725)×0.007184. In some embodiments, a patient's body surface area is calculated according to the following formula:

${{BSA}\mspace{11mu}\left( m^{2} \right)} = {\sqrt{\frac{\left\lbrack {{height}\mspace{14mu}({cm}) \times {weight}\mspace{14mu}({kg})} \right.}{3600}}.}$

In some embodiments, the disclosure provides methods of coadministering everolimus and CBD (e.g., having a purity of at least 95% or 98% w/w) to treat TSC patients with moderate or severe hepatic impairment. In some embodiments, such patients may receive a lower starting dose, maintenance dose, and maximum dose compared to the dose and otherwise identical patient with moderate or severe hepatic impairment would receive in the absence of everolimus. Table 3 lists CBD doses for patients with moderate or severe hepatic impairment in the absence of everolimus.

TABLE 3 CBD Doses Patients with Hepatic Impairment Maximum Hepatic Recommended Impairment Starting Dosage Dosage Mild 2.5 mg/kg twice daily 12.5 mg/kg twice (5 mg/kg/day) daily (25 mg/kg/day) Moderate 1.25 mg/kg twice 6.25 mg/kg twice daily (2.5 mg/kg/day) daily (12.5 mg/kg/day) Severe 0.5 mg/kg twice daily 2.5 mg/kg twice daily (1 mg/kg/day) (5 mg/kg/day)

In some embodiments, a TSC patient with mild renal impairment is co-administered 2.5 or 5 mg everolimus, and a staring dose of CBD that is less than 5 mg/kg/day (e.g., about 1, about 2, about 3, about 4, or about 5 mg/kg/day). In some embodiments, the starting dose is increased by about 1 mg/kg/day to about 5 mg/kg/day (e.g., about 1, about 2, about 3, about 4, or about 5 mg/kg/day) up to a maximum dose of less than 25 mg/kg/day (e.g., about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, mg/kg/day, including all values and ranges between these values). In some embodiments, patients with mild hepatic impairment are treated with a dose of CBD ranging from about 5 to about 20 mg/kg/day.

In some embodiments, a TSC patient with moderate renal impairment is co-administered 2.5 or 5 mg everolimus, and a staring dose of CBD that is less than 2.5 mg/kg/day (e.g., about 0.5 mg/kg/day, or about 1 mg/kg/day). In some embodiments, the starting dose is increased by about 0.5 mg/kg/day to about 2.5 mg/kg/day (e.g., about 0.5, about 1, about 1.5 or about 2 mg/kg/day) up to a maximum dose of less than 12.5 mg/kg/day (e.g., about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2.5 mg/kg/day, including all values and ranges between these values). In some embodiments, patients with moderate hepatic impairment are treated with a dose of CBD ranging from about 2.5 to about 10 mg/kg/day.

In some embodiments, a TSC patient with severe renal impairment is co-administered 2.5 or 5 mg everolimus, and a staring dose of CBD that is less than 1 mg/kg/day (e.g., about 0.25, mg/kg/day, or about 0.5 mg/kg/day). In some embodiments, the starting dose is increased by about 0.5 mg/kg/day up to a maximum dose of less than 5 mgkg/day (e.g., about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or 4.5 mg/kg/day).

In some embodiments, the patent had a diagnosis of TSC and the seizures were inadequately controlled with at least one concomitant anti-epileptic drug (AED), with or without vagal nerve stimulation or ketogenic diet. In some embodiments, the AED is valproate, vigabatrin, levetiracetam, clobazam, or combinations thereof.

EXAMPLES Example 1. Preparation of a CBD Extract

A highly purified (>98% w/w CBD) cannabidiol extract was prepared for treatment of TSC patients. The production of this extract is also described in U.S. Pat. No. 10,111,840 and U.S. Patent App. Pub. No. 2016/0166514, each of which are incorporated by reference herein in its entirety.

Summary: CBD was extracted from high-CBD containing chemotypes of Cannabis sativa L. using a liquid carbon dioxide extraction method. The CBD extract was further purified using a solvent crystallization method to yield CBD. The crystallization method specifically removes other cannabinoids to yield a CBD extract that contains greater than 95% w/w CBD and typically greater than 98% w/w CBD.

Procedure: The Cannabis sativa L. plants are grown, harvested, and processed to make a botanical extract (intermediate). The plant starting material is referred to as Botanical Raw Material (BRM). The active pharmaceutical ingredient (API), also called drug substance, is CBD.

The BRM and botanical extract (intermediate) are controlled by the specifications. The drug substance specification is described in Table 4.

TABLE 4 CBD Specification Test Test Method Limits Apperance Visual Off-white/pale yellow crystals Identification A HPLC-UV Retention time of major peak corresponds to certified CBD reference standard Identification B GC-FID/MS Retention time and mass spectrum of major peak corresponds to certified CBD reference standard Identification C FT-IR Conforms to reference spectrum for certified CBD reference standard Identification D Melting Point 65° C. to 67° C. Identification E Specific Optical Rotation Conforms with certified CBD Reference Standard; −110° to −140° (in 95% ethanol) Total Purity Calculation ≥98.0 % Chromatographic Purity 1 High Performance Liquid ≥98.0 % Chromatography - Ultraviolet (UV) Chromatographic Purity 2 Gas Chromatography-flame >98.0 % ionization detector/mass spectrometry (GC-FID/MS) CBDA High Performance Liquid Not more than 0.15 % w/w Chromatography - UV CBDV High Performance Liquid Not more than 1.0 % w/w Chromatography - UV A9THC High Performance Liquid Not more than 0.15 % w/w Chromatography - UV CBD-C4 High Performance Liquid Not more than 0.5 % w/w Chromatography - UV Alkane Gas chromatography Not more than 0.5 % w/w Ethanol Gas chromatography Not more than 0.5 % w/w Residual Water Karl Fischer Not more than 1.0 % w/w

The purity of the CBD drug substance achieved is greater than 98%. The other cannabinoids which may occur in the extract are: CBDA, CBDV, CBD-C4 and THC.

Distinct chemotypes of Cannabis sativa L. plant have been produced to maximize the output of the specific chemical constituents, the cannabinoids. One type of plant produces predominantly CBD. Only the (−)-trans isomer occurs naturally, furthermore during purification the stereochemistry of CBD is not affected.

An overview of the steps to produce a botanical extract, the intermediate, are as follows:

(i) Growing

(ii) Decarboxylation

(iii) Extraction Number 1: Using liquid CO₂

(iv) Extraction Number 2: “Winterization” using ethanol

(v) Filtration

(vi) Evaporation

High CBD chemovars were grown, harvested and dried and stored in a dry room until required. The botanical raw material (BRM) was finely chopped using an Apex mill fitted with a 1 mm screen. The milled BRM was stored in a freezer for up to 3 months prior to extraction. Decarboxylation of CBDA to CBD was carried out using a large Heraeus tray oven.

The decarboxylation batch size in the Heraeus is approximately 15 Kg. Trays were placed in the oven and heated to 105° C.; the BRM took 96.25 minutes to reach 105° C. Held at 105° C. for 15 Minutes. Oven then set to 150° C.; the BRM took 75.7 minutes to reach 150° C.; BRM held at 150° C. for 130 Minutes. Total time in the oven was 380 Minutes, including 45 minutes cooling and 15 Minutes venting.

Extraction Number 1 was performed using liquid CO₂ at 60 bar/10° C. to produce botanical drug substance (BDS) which was used for crystallisation to produce the test material.

The crude CBD BDS was winterized in Extraction Number 2 under standard conditions (two volumes of ethanol at minus 20° C. for around 50 hours). The precipitated waxes were removed by filtration.

The manufacturing steps to produce the drug substance (CBD) from the intermediate botanical extract are as follows:

-   -   (i) Crystallization using C5-C12 straight chain or branched         alkane     -   (ii) Filtration     -   (iii) Optional recrystallization from C5-C12 straight chain or         branched alkane     -   (iv) Vacuum Drying

Intermediate botanical extract (12 kg) produced using the methodology above was dispersed in C5-C12 straight chain or branched alkane (9000 ml, 0.75 vols) in a 30 liter stainless steel vessel. The mixture was manually agitated to break up any lumps and the sealed container then placed in a freezer for approximately 48 hours.

The crystals were isolated by vacuum filtration, washed with aliquots of cold C5-C12 straight chain or branched alkane (total 12000 ml), and dried under a vacuum of <10 mb at a temperature of 60° C. until dry before submitting the drug substance for analysis. The dried product was stored in a freezer at minus 20° c. in a pharmaceutical grade stainless steel container, with FDA food grade approved silicone seal and clamps.

The drug product is presented as an oral solution containing the drug substance. The oral solution presentation contains 25 mg/ml or 100 mg/ml CBD, with the excipients sesame oil, ethanol, sucralose and flavoring. Two product strengths are available to allow dose titration across a wide dose range. The 25 mg/ml solution is appropriate at lower doses and the 100 mg/ml solution at higher doses.

The drug product is described in Table 5 below.

TABLE 5 Drug Product Specification Reference Qualitative to Quality Component Composition Function Standard Cannabidiol (CBD)   25 mg/mL or Active Ingredient In-house  100 mg/mL Anhydrous Ethanol 79.0 mg/mL Excipient Ph.Eur. Sucralose  0.5 mg/mL Sweetener In-house Strawberry flavoring  0.2 mg/mL Flavoring In-house Sesame Oil q.s. to 1.0 mL Excipient Ph.Eur.

The drug substance CBD is insoluble in water. Sesame oil was selected as an excipient to solubilize the drug substance. A sweetener and fruit flavoring are required to improve palatability of the sesame oil solution. Ethanol was required to solubilize the sweetener and the flavoring. The composition can be substantially equivalent, by which is meant that the function ingredients can vary from the qualitative composition specified in Table 6 by an amount up to 10%.

Example 2. Inhibition of CYP450 by CBD

In vitro studies were performed to measure CYP450 inhibition by CBD. CBD has an IC₅₀=1.42 μM against CYP3A4-mediated testosterone hydroxylation. CBD also has a K_(I)=1.5 against CYP3A4-mediated testosterone hydroxylation, K_(inact)=0.14 min′.

The in vitro data shows that CBD has the potential to inhibit multiple CYP450s in vivo.

While the in vitro data suggest that CBD may cause DDI with CYP3A4 substrates, clinical DDI study with midazolam (GWEP17028, herein incorporated by reference) showed no clinically significant effect on orally dosed midazolam PK parameters when dosed concomitantly with CBD at steady-state (750 mg bid, fed state).

Example 3. A Phase 1, Open-Label, Pharmacokinetic Drug-Drug Interaction Trial to Investigate the Effect of Cannabidiol on the Pharmacokinetics (PK) of Everolimus

Rationale: Everolimus is an mTOR inhibitor approved as an oral treatment in patients with tuberous sclerosis complex (TSC) to manage TSC associated-seizures, where it may be used concomitantly with CBD. Nonclinical ADME data show that there is potential for CBD to interact with the pathways involved in the absorption and elimination of everolimus. The principal aim of this trial was to assess the effects that CBD has on the pharmacokinetic (PK) parameters of everolimus.

Primary Objectives: To investigate the effect of CBD treatment following repeated dosing on the PK of a single dose of everolimus in healthy participants.

Primary Endpoints: The PK parameter endpoints, derived from the plasma concentration-time profiles of everolimus on Day 1 administered alone and the PK parameter endpoints derived from a single dose of everolimus in participants at steady state CBD following 5 days of CBD, 12.5 mg/kg twice daily (b.i.d).

Secondary Objectives: To evaluate the safety and tolerability of CBD when given with a single dose of everolimus in healthy participants; to investigate the effect of a single everolimus dose on the PK parameters of CBD at steady state in healthy participants

Secondary Endpoints: Safety includes: incidence and severity of adverse events (AEs), incidence of laboratory abnormalities based on hematology, clinical chemistry, and urinalysis test results; 12-lead electrocardiogram (ECG) parameters, vital sign measurements, physical examinations, Columbia-Suicide Severity Rating Scale (C-SSRS) questionnaire scores; the PK parameter endpoints, derived from the plasma concentration-time profiles of everolimus on Day 1 administered alone and the PK parameter endpoints derived from a single dose of everolimus in participants at steady state CBD following 5 days of CBD, 12.5 mg/kg twice daily (b.i.d) (total daily dose of 25 mg/kg/day).

Exploratory Objectives: To investigate potential effects of CYP3A genotypes on everolimus exposure and any interaction with CBD.

Exploratory Endpoints: pharmacogenetic assessment of common genetic variants in CYP3A enzymes (this information may be correlated with PK outcomes)

Design: This was a phase 1, open-label, single site trial to investigate the effect of multiple dose administration of CBD on the PK of everolimus in healthy participants. The duration of the trial was approximately 9 weeks, which includes a screening period (up to 4 weeks), a treatment period (18 days) and a safety follow-up period (2 weeks). After signing the informed consent form (ICF), participants enter the screening period (Day −28 to −1). On Day −1, screened participants who continued to meet eligibility criteria were admitted to the clinical research unit (CRU) at approximately 2 pm and enrolled into the trial. On Day 1, participants received everolimus, 30 minutes after starting a standard meal. On Days 2 to 8, participants had a 7-day washout period. On Days 9 to 12, participants received CBD, 30 minutes after starting a standard meal. On Day 13, participants received everolimus and CBD, 30 minutes after starting a standard meal. On Days 14 to 18, participants received CBD, 30 minutes after starting a standard meal. On Day 18, participants were discharged from the CRU once all assessments were completed. On Day 32, a safety follow-up visit was conducted 14 days (±3 days) after the last dose of investigational medicinal product (IMP), CBD.

During the initial trial, patients received 5 mg everolimus and 12.5 mg/kg CBD b.i.d. (total daily dose of 25 mg/kg/day).

Many doses of CBD and everolimus are tested according to the study design outlined above, as shown in Table 6. CBD is administered twice daily at the doses listed in Table 6.

TABLE 6 Potential Doses of CBD (b.i.d.) and everolimus Everolimus CBD 2.0 mg 2.5 mg/kg 2.0 mg 5 mg/kg 2.0 mg 10 mg/kg 2.0 mg 12.5 mg/kg 2.5 mg 2.5 mg/kg 2.5 mg 5 mg/kg 2.5 mg 10 mg/kg 2.5 mg 12.5 mg/kg 5 mg 2.5 mg/kg 5 mg 5 mg/kg 5 mg 10 mg/kg 5 mg 12.5 mg/kg 10 mg 2.5 mg/kg 10 mg 5 mg/kg 10 mg 10 mg/kg 10 mg 12.5 mg/kg 4.5 mg/m² 2.5 mg/kg 4.5 mg/m² 5 mg/kg 4.5 mg/m² 10 mg/kg 4.5 mg/m² 12.5 mg/kg 2.25 mg/m² 2.5 mg/kg 2.25 mg/m² 5 mg/kg 2.25 mg/m² 10 mg/kg 2.25 mg/m² 12.5 mg/kg 5.0 mg/m² 2.5 mg/kg 5.0 mg/m² 5 mg/kg 5.0 mg/m² 10 mg/kg 5.0 mg/m² 12.5 mg/kg 2.5 mg/m² 2.5 mg/kg 2.5 mg/m² 5 mg/kg 2.5 mg/m² 10 mg/kg 2.5 mg/m² 12.5 mg/kg

Formulation Mode of Administration, Dose, Regimen: The BCD formulation is an oral liquid formulation that is clear and colorless to yellow in appearance (100 mg/mL CBD in sesame oil with anhydrous ethanol, added sweetener (sucralose), and strawberry flavoring. The oral liquid formulation is administered with a syringe. The CBD formulation was taken b.i.d. 30 minutes after starting a standard meal

Everolimus was taken by mouth (p.o) 30 minutes after a standard meal.

Procedures: All visits were scheduled to take place at approximately the same time of day (i.e., morning or afternoon), whenever possible. Cannabis or benzodiazepines will not be allowed throughout the trial. Alcohol was not allowed from 48 hours prior to each admission to the CRU and throughout the inpatient period, and from 48 hours prior to the safety follow-up visit.

Pharmacokinetic Assessments: PK blood sampling was collected for everolimus and CBD, 7-hydroxy-cannabidiol (7-OH-CBD) and 7-carboxy-cannabidiol (7-COOH-CBD) and were collected during the treatment period at the timepoints listed in the Schedule of Assessments. The time of the meal consumed at the site during days of IMP administration was recorded, as was the dose and dosing time of IMP (as appropriate) relative to sampling times.

The following assessments were performed: demographics, medical history, physical examination, vital signs, body weight, height, 12-lead ECG, adverse events (AEs), previous and concomitant medications recorded. The investigator or delegate completes the C-SSRS. Clinical laboratory samples including chemistry, hematology, serology, urine drug screen, alcohol test, and pharmacogenetics.

Treatment Period Assessments: Participants who continued to satisfy all inclusion criteria and none of the exclusion criteria, were enrolled to receive everolimus and CBD. The following assessments were performed: physical examination, vital signs, 12-lead ECG, concomitant medications recorded, and AEs are reviewed. The investigator or delegate completes the C-SSRS. Clinical laboratory samples including chemistry, coagulation, hematology, and samples for PK analysis.

Safety Follow-Up Visit Assessments: The following assessments were performed: physical examination, vital signs, 12-lead ECG, adverse events (AEs), and concomitant medications recorded. The investigator or delegate completed the C-SSRS. Clinical laboratory samples including chemistry and hematology.

Pharmacokinetic parameter estimates were evaluated to assess the change in PK parameters of CBD, 7-OH-CBD and 7-COOH-CBD at steady state CBD, following a single dose of everolimus.

Log transformed C_(max), AUC_((0-∞)), and AUC_((0-t)) parameters for everolimus were analyzed using a linear mixed-effects model with a fixed effect for treatment and a random effect for participant. The treatment differences are back transformed to present the ratios of geometric means and the corresponding 90% confidence intervals (CIs).

For t_(max), nonparametric analysis of the same comparisons were performed using a Wilcoxon signed-rank test. Medians and median differences between the treatments were presented along with the approximate 90% CI for the median difference.

Geometric mean ratios and 90% CIs were used to estimate the magnitude of any interaction and will be interpreted based on clinical relevance.

G_(trough) values for CBD, 7-OH-CBD and 7-COOH-CBD are listed and summarized as appropriate.

All AEs are listed and summarized using descriptive methodology. The incidence of AEs for each treatment period are presented by severity and by association with the IMPs as determined by the Investigator (or designee). All safety data is listed and summarized using a descriptive methodology. Pharmacogenetic data will be listed.

Results:

Initially, patients received 5 mg everolimus, or 5 mg everolimus and 12.5 mg/kg CBD b.i.d. (total daily dose of 25 mg/kg/day) according to the trial design described above. See FIG. 1. 5 mg everolimus and 25 mg/kg/day CBD represent the dose of everolimus and CBD, respectively, that TSC patients would receive based on the current FDA labels.

The blood plasma concentration profiles of everolimus and CBD were measured the time points described above. FIG. 2 shows the blood concentration profile (ng/mL) of everolimus in patients that received everolimus alone, and patients that received both everolimus and CBD. As shown in FIG. 2, CBD surprisingly and unexpectedly causes elevations in the blood plasma concentrations of everolimus, even though CBD was not considered to be moderate or strong CYP3A4 and P-gp inhibitor. The results are summarized in Table 7.

TABLE 7 Analysis of Drug-Drug Interaction PK Everolimus (EVE) Geometric LS means EVE EVE + PK Alone CBD Ratio Test/Reference Analyte Parameter (n = 16) (n = 16) Estimate 91% CI (lower, upper) P value Everolimus Cmax  18  46 2.5 (2.12, 2.94) <0.0001 (ng/mL) AUC_(0-last)  95 497 2.55 (2.23, 2.90) <0.0001 (h*ng/mL) AUC_(0-inf) 216 529 2.45 (2.15, 2.80) <0.0001 (h*ng/mL)

As shown above in Table 7 and FIG. 2, CBD (at SS) increased the Cmax and AUC of everolimus by at least 2.5-fold. This is a clinically significant, moderate interaction (Clinical Drug Interaction Studies—Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry, FDA, January 2020). Consequently, the dose of CBD was reduced (e.g., as described herein) to mitigate the DDI with everolimus.

FIG. 3-5 shows that everolimus did not have a clinical significant impact of the pharmacokinetics of CBD, or its metaboliates 7-OH-CBD, and 7-COOH-CBD, respectively.

As noted above, the CBD (at SS) increased the Cmax and AUC of everolimus by at least 2.5-fold. In order to mitigate the impact of CBD on everlimus blood plasma concentration, and reduce the risk of side effects related to everolimus overexposure, the dose of CBD administered to TSC is reduced as described herein. It is important to note that reducing the CBD dose by 2.5 fold is not required to safely and effectively co-administer everolimus and CBD (although a 2.5 fold reduction in the CBD is encompassed by this disclosure). In some embodiments, the CBD dose need only be reduce enough such that the everolimus AUC (or Cmax) is increased by no more than 2 fold. Without being bound by theory, this can be achieved by administering 20 mg/kg/day CBD in combination with everolimus, as less than 2 fold increases in everolimus AUC (or Cmax) are not considered clinically relevant.

In another example, to mitigate the impact of CBD on everlimus blood plasma concentration, and reduce the risk of side effects related to everolimus overexposure, the dose of everolimus is reduced as described herein. It is important to note that reducing the everolimus dose by 2.5 fold is not required to safely and effectively co-administer everolimus and CBD (although a 2.5 fold reduction in the CBD is encompassed by this disclosure). In some embodiments, the CBD dose need only be reduce enough such that the everolimus AUC (or Cmax) is increased by no more than 2 fold. Without being bound by theory, this can be achieved by reducing the everolimus dose by 10%, 15%, 20%, and/or 25%.

Numbered Embodiments of the Disclosure

Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

Embodiment 1. A method of treating seizures associated with tuberous sclerosis complex (TSC) in a patient in need thereof, comprising administering everolimus in combination with a reduced dose of cannabidiol (CBD).

Embodiment 2. The method of embodiment 1, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day.

Embodiment 3. The method of embodiment 1 or 2, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 4. The method of embodiment 1 or 2, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 5. The method of embodiment 1 or 2, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 15 mg/kg/day.

Embodiment 6. The method of embodiment 1 or 2, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 20 mg/kg/day.

Embodiment 7. The method of embodiment 1, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, and about 15 mg/kg/day.

Embodiment 8. The method of embodiment 1 or 7, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 9. The method of embodiment 1 or 7, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 10. The method of embodiment 1 or 7, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 15 mg/kg/day.

Embodiment 11. The method of embodiment 1, wherein the reference dose of CBD is 15 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, and about 10 mg/kg/day.

Embodiment 12. The method of embodiment 1 or 11, wherein the reference dose of CBD is 15 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 13. The method of embodiment 1 or 11, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 14. The method of embodiment 1 or 7, wherein the reference dose of CBD is 10 mg/kg/day, and the patient is administered 2.5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 15. The method of embodiment 1, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, and about 20 mg/kg/day.

Embodiment 16. The method of embodiment 1 or 15, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 17. The method of embodiment 1 or 15, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 18. The method of embodiment 1 or 15, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 15 mg/kg/day.

Embodiment 19. The method of embodiment 1 or 15, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 20 mg/kg/day.

Embodiment 20. The method of embodiment 1, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, about 10 mg/kg/day, and about 15 mg/kg/day.

Embodiment 21. The method of embodiment 1 or 20, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 22. The method of embodiment 1 or 20, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 23. The method of embodiment 1 or 20, wherein the reference dose of CBD is 20 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 15 mg/kg/day.

Embodiment 24. The method of embodiment 1, wherein the reference dose of CBD is 15 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is selected from the group consisting of about 5 mg/kg/day, and about 10 mg/kg/day.

Embodiment 25. The method of embodiment 1 or 24, wherein the reference dose of CBD is 15 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

Embodiment 26. The method of embodiment 1 or 24, wherein the reference dose of CBD is 25 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 10 mg/kg/day.

Embodiment 27. The method of embodiment 1 or 15, wherein the reference dose of CBD is 10 mg/kg/day, and the patient is administered 5 mg of everolimus, and the reduced dose of CBD is about 5 mg/kg/day.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. 

1-43. (canceled)
 44. A method of treating seizures associated with tuberous sclerosis complex in an everolimus-naive patient, wherein the patient is currently treated with CBD, comprising: (a) coadministering everolimus to the patient; (b) monitoring the patient's blood plasma concentrations of everolimus; and (c) if the patient's blood plasma trough concentrations of everolimus exceed 15 ng/mL, then reducing the dose of everolimus by at least 10%.
 45. The method of claim 44, wherein the patient is treated with 25 mg/kg/day CBD.
 46. The method of claim 44, wherein step (a) comprises administering 5 mg/m² everolimus once daily.
 47. The method of claim 46, wherein the dose of everolimus is titrated up by no more than 5 mg until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL.
 48. The method of claim 47, wherein the dose of everolimus is titrated every 1 to 2 weeks until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL.
 49. The method of claim 44, wherein the monitoring in step (b) occurs 1 to 2 weeks after the patients begins administering everolimus.
 50. A method of treating seizures associated with tuberous sclerosis complex in an everolimus-naive patient, wherein the patient is currently treated with CBD, comprising: (a) administering everolimus to the patient; (b) monitoring the patient's blood plasma concentrations of everolimus; and (c) if the patient's Cmax exceeds 50 ng/mL or AUC₀₋₂₄ 500 h*ng/mL, then reducing the dose of everolimus by at least 10%.
 51. The method of claim 50, wherein the patient is treated with 25 mg/kg/day CBD.
 52. The method of claim 50, wherein step (a) comprises administering 5 mg/m² everolimus once daily.
 53. The method of claim 52, wherein the dose of everolimus is titrated up by no more than 5 mg until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL.
 54. The method of claim 53, wherein the dose of everolimus is titrated every 1 to 2 weeks until the patient's blood plasma trough concentration of everolimus is within the range of 5-15 ng/mL.
 55. The method of claim 50, wherein the monitoring in step (b) occurs 1 to 2 weeks after the patient begins administering everolimus.
 56. The method of claim 44, wherein the dose of everolimus is reduced by at least 10%.
 57. The method of claim 56, wherein the dose of everolimus is reduced to provide a blood plasma trough concentration of everolimus that is within the range of 5-15 ng/mL.
 58. The method of claim 56, wherein the dose of everolimus is reduced to provide a steady state Cmax that is not more than about 45 ng/mL.
 59. The method of claim 56, wherein the dose of everolimus is reduced to provide an steady state AUC₀₋₂₄ that is not more than about 490 h*ng/mL. 